Abstract
Manumycin A is postulated to be a specific inhibitor against the farnesyltransferase (FTase) since this effect has been shown in 1993 for yeast FTase. Since then, plenty of studies investigated Manumycin A in human cells as well as in model organisms like Caenorhabditis elegans. Some studies pointed to additional targets and pathways involved in Manumycin A effects like apoptosis. Therefore, these studies created doubt whether the main mechanism of action of Manumycin A is FTase inhibition. For some of these alternative targets half maximal inhibitory concentrations (IC50) of Manumycin A are available, but not for human and C. elegans FTase. So, we aimed to 1) characterize missing C. elegans FTase kinetics, 2) elucidate the IC50 and Ki values of Manumycin A on purified human and C. elegans FTase 3) investigate Manumycin A dependent expression of FTase and apoptosis genes in C. elegans. C. elegans FTase has its temperature optimum at 40°C with KM of 1.3 µM (farnesylpyrophosphate) and 1.7 µM (protein derivate). Whilst other targets are inhibitable by Manumycin A at the nanomolar level, we found that Manumycin A inhibits cell-free FTase in micromolar concentrations (Ki human 4.15 μM; KiC. elegans 3.16 μM). Furthermore, our gene expression results correlate with other studies indicating that thioredoxin reductase 1 is the main target of Manumycin A. According to our results, the ability of Manumycin A to inhibit the FTase at the micromolar level is rather neglectable for its cellular effects, so we postulate that the classification as a specific FTase inhibitor is no longer valid.
Introduction
Historically, Manumycin A was developed as an antimicrobial agent isolated from Streptomyces pavulus Tu64 (). In 1993, it was described as a highly potent, specific farnesyltransferase (FTase) inhibitor (FTI) in yeast (). The inhibitory effect seems to be based on its analogy to the prenyl substrate farnesylpyrophosphate ().
FTase belongs to the group of prenyltransferases (FTase and geranylgeranyltransferase I-III (GGTase I-III)) (; ; ), performing an important post-translational modification in eukaryotes. Canonically, prenylation leads to membrane association and functionality of about 200 proteins in cell signalling pathways (; ). FTase catalyses the post-translational attachment of a farnesyl (15C) moiety to the cysteine of the so called CAAX-box at the C-terminus of proteins (; ; ; ). Well-known farnesylated proteins are the small GTPases of the Ras-superfamily including Rab, Ran, Ras, Rho and Arf, influencing cellular processes like cell differentiation and inflammation (). Members of the Ras-family are known to play a crucial role in tumorgenesis, explaining the extensive research on the drug class of FTIs and its member Manumycin A (; ).
Most of the Manumycin A experiments have been carried out in cell cultures, as well as on C. elegans. Here, an age dependent reduction in motility was observed. Additionally, Manumycin A evoked toxic effects on the worm, which are supposed to be due to the farnesyltransferase inhibiting properties of Manumycin A (; ).
Remarkably, a lot of publications describe effects of Manumycin A on additional targets in human cells. For example, Manumycin A inhibits Iκ-B kinase β or the thioredoxin-reductase 1 (; ). Manumycin A also seems to have an influence on phospho-protein kinase B (pAKT), PI3K (phosphoinositol 3 -kinase) and specificity protein 1 (Sp1) (; ). Several studies showed that Manumycin A is effective against different cancer types () e.g., prostate cancer. Here, Manumycin A induced apoptosis in LNCaP cells, which was based on the intrinsic activation of Caspase-9 and -3 (Cas-9, Cas-3). Additionally, an upregulation of B-cell lymphoma 2 (Bcl-2) and a downregulation of Bcl-2 associated X protein (Bax) could be observed. Besides these pro-apoptotic effects, Manumycin A seems to have antiatherosclerotic, antibacterial, anti-inflammatory, antimycotic, anti-neurogenerative and hepatoprotective properties (; ; ; ). All these findings create doubt that Manumycin A is just an FTI. So, we analyzed the postulated role of Manumycin A as a farnesyltransferase inhibitor and determined its kinetic parameters. Additionally, we reviewed the different effects described and postulate a new main way of action for Manumycin A explaining all previous findings.
Materials and methods
Cloning and heterologous expression in Escherichia coli
The Escherichia coli strains DH5α (Thermo Fisher Scientific, Waltham, MA, USA), and Rosetta (DE3)pLysS (Novagen, Darmstadt, Germany) for cloning and expression studies were cultured under standard conditions following the instructions of the manufacturers. The cloning of the coding regions of C. elegans FTα (ceFNTA, NM_001392242.1) and FTβ (ceFNTB, NM_001392654.1) in pETDuet1 (Novagen) was performed by BioCat (Mannheim, Germany). pETDuet1 harbors two multiple cloning sites with one comprising a 6xHIS-tag. In both co-expression plasmids, the β-subunit is cloned in frame with an N-terminal His-tag. Cells were grown in LB-medium at 37°C containing 50 µg/ml ampicillin. Expression was induced by addition of 0.4 mM isopropyl-1-thio-β-D-galactopyranoside at an OD600 of ∼ 0.6 and 0.5 mM ZnSO4. After induction cells were grown for 4 h at 34°C (), harvested by centrifugation (8,000×g, 4°C, 20 min) and stored at -80°C.
Purification of recombinant enzymes
Affinity chromatography with Ni-NTA was used to co-purify His-tagged FTβ together with FTα. E. coli cells were suspended (5 ml/1 g wet cell weight) in buffer A (50 mM Tris/HCl, pH 7.5, 300 mM KCl, 10 mM MgCl2, 10 µM ZnCl2, 20 mM imidazole, 5 mM DTT) and protease inhibitor cocktail (Roche, Basel, Switzerland) and lysed by sonification (3 × 5 min) in an ice ethanol bath. Cell debris and intact cells were removed by centrifugation (21100 x g, 45 min, 4°C). The lysate was applied to a Ni-NTA IMAC (immobilized metal ion affinity chromatography) column (Qiagen, Hilden, Germany) equilibrated with 3 × 5 ml of buffer A. The flow through was applied five times to the column by gravity flow. After washing (3 × 10 ml buffer A by gravity flow) the column was incubated on ice for 5 min with 200 µl buffer AE (buffer A, 250 mM imidazole) and eluted by gravity flow.
Immuno-blot and -detection
Protein fractions were analyzed by SDS-PAGE and infra-red immuno blot (LiCor, Lincoln, NE, USA) with a coupled specific His-infra-red antibody (6x-His-tag antibody, 1:2000, DyLight 680, Thermo Fisher Scientific, Waltham, MA, USA). After semi-dry blotting (30 min, 25 V) the nitrocellulose membrane was blocked (RT, 60 min, shaking) in blocking solution (LiCor), incubated with His-infra-red antibody (1:2000, blocking solution, 0.1% Tween20, RT, 60 min, shaking), washed (3 × 5 min, PBS, 0.1% Tween20, and 1 × 5 min PBS), dried in the dark and visualized on an Odyssey imager (LiCor).
Continuous fluorescence assay
Enzyme activity of FTase was analyzed using a continuous fluorescence assay (). The assay was performed with enzyme purified via IMAC in black flat 96-well plates (Thermo Scientific) with a total volume of 250 µL.
Different concentrations (see determination of Km-/IC50-values) of peptide substrate Dansyl-GCVLS were preincubated with 5 mM DTT in H2O (total volume 50 µL) for 30 min at room temperature. Preincubated Dansyl-GCVLS/DTT solution and 120 µL of enzyme solution (2 µg enzyme in H2O) were incubated in assay buffer (final buffer concentrations of 50 mM Tris-HCl pH 7.5, 10 µM ZnCl2 and 0.03% n-Dodecyl-ß-D maltoside) for 3 min at 30°C. The reaction was started by adding 30 µL of farnesylpyrophosphate (FPP)/H2O solution in varying FPP concentrations (see determination of Km-/IC50-values). The increase in fluorescence was measured every 30 s for 60 min (extinction: 340nm, emission: 505 nm). All measurements of human FTase were performed at 30°C. For the determination of the optimal reaction temperature of C. elegans FTase, the enzyme was measured at varying temperatures using a Tecan microplate reader (Tecan Infinite 2000 PRO mPlex, Männedorf, Tecan group Ltd., Switzerland) from 7–40°C. Since the readers maximal temperature is 40°C, we performed heat precipitation with Ni-NTA purified enzyme. Therefore, ceFTase was incubated at 45°C and 50°C, respectively for 20 min and afterwards centrifuged for 30 min at 16000xg. The soluble fractions were measured as described and analyzed with the resulting pellet fractions by an SDS-PAGE.
Determination of apparant KM-values
For the determination of the apparant KM-value for Dansyl-GCVLS, the concentration of the substrate was varied (0.125–16 µM) while the concentration of FPP was kept constant at a saturating substrate concentration of 10 µM. Accordingly, for the KM of FPP, concentration of FPP was varied (0.125–16 µM), while the concentration of Dansyl-GCVLS was constant at 8 µM.
Determination of IC50-values
Furthermore, half-maximal inhibitory concentrations were determined for the inhibitor Manumycin A with the continuous fluorescence assay at the optimal temperatures of the responding enzyme (30°C for human and 40°C for ceFTase). Substrate concentration of Dansyl-GCVLS (8 µM) and FPP (2 µM) were constant. For ceFTase Manumycin A was tested at final concentrations of 0.1–1,000 µM. With respect to hFTase, Manumycin A was tested at final concentrations of 0.1–200 µM. Manumycin A was dissolved in DMSO; the final concentration of DMSO did not exceed 5% in the assay. The initial reaction rates from the series of enzyme assays were calculated at varying inhibitor concentrations and related to the reaction rate in absence of inhibitor. The residual enzyme activity was plotted against the inhibitor concentration. IC50 was analyzed with GraphPad Prism (Non-linear regression, Dose-Response Inhibition). Experiments were conducted three times each (n = 3).
Cell culture
LNCaP and PC3 cells are human prostate adenocarcinoma cells. HEK 293 are human embryo kidney cells. All cell lines were kindly provided from the Institute of Pharmacogenetics, University Hospital Essen, Essen, Germany. Cell line genotyping determined the identity of the cell lines. LNCaP were cultured in RPMI, HEK 293 in DMEM and PC3 in 50% RPMI and 50% Ham´s F-12, all media contained 10% FBS and 100 units/ml Penicillin/Streptomycin (1%) in a humidified atmosphere at 37°C and 5% CO2. Cells were passaged every 3–4 days at a confluence of 90%.
Cell viability assay (MTT)
For analysis of the cell viability, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay (Merck Millipore, Burlington, Massachusetts, USA) was used. In a 96-well plate, 5,000 (LNCaP), 10,000 (PC3) or 15,000 (HEK293) cells per well were seeded and grown for 24 h. Then cells were incubated with Manumycin A in different concentrations (1–60 µM) for 48 h. After incubation, MTT assay was performed according to manufacturer`s protocol.
Cultivation of C. elegans
Strain maintenance was performed under standard conditions on Nematode Growth Medium (NGM) agar as described by (). E. coli OP50 streaked on the plates was used as feeding source. The wild-type strain N2 was used after three to four generations. The treatment with Manumycin A and DMSO respectively was performed as follows: feeding plates with 20 ml media were plated, inoculated and incubated with E. coli OP50 and afterwards coated with 3 μM, 9 μM, 25 μM, 50 μM or 100 μM Manumycin A as final concentration. The highest amount (0.55%) of DMSO was applied to plates, serving as negative control. As an additional control, the TrxR inhibitor D-9 was applied to the plates (300 nM, 100 nM, 30 nM and 10 nM). After drying, 1-3 worms were put on the plates, incubated for 6–7 days at 22°C, harvested and washed with phosphate buffered saline (PBS).
For the measurement of endogenous FTase from C. elegans, plates and worms were prepared as described above. To test for FTase inhibition by Manumycin A, 63 nM were applied to the plates; this is the concentration where a phenotype, but no toxicity was observed by Bar and Gruenbaum (). Additionally, we cultivated non-treated as well as DMSO-treated (0.55%) C. elegans. Worms were harvested from ∼30 plates using PBS and washed in Lysis-Buffer (50 mM Tris/HCl, pH 7.5, 300 mM KCl, 10 mM MgCl2, 10 µM ZnCl2, 20 mM imidazole, 5 mM DTT and protease inhibitor cocktail (Roche, Basel, Switzerland)). After three freeze thaw cycles with liquid nitrogen and a 37°C water bath, the worms were mechanically lysed in a homogeniser and centrifuged (4°C, 16.000 x g, 45 min) (). The supernatant was used in the continuous fluorescent assay as described above. We determined the initial flow rate using GraphPad Prism Software (GraphPad Software 8.0, San Diego, CA, USA).
RNA extraction and cDNA synthesis
C. elegans were washed from the plate harvested in 1 ml PBS and stored at -80°C overnight. After thawing, samples were homogenized using a rotor-stator homogenizer (TissueLyser II, Qiagen, Hilden) and total RNA preparation was performed using the QuickRNA-Kit (Zymo Research, Freiburg, Germany) according to the manufacturer’s instructions. RNA was quantified photometrically at 260 and 280 nm using a Tecan Infinite200 PRO plate reader. An OD260nm/280nm of 1.9–2.1 was considered as protein free RNA (Supplementary Table S1). All RNA samples were stored at -80°C.
For cDNA synthesis, we used the PrimeScript RT Master Mix (Takara Bio, Kusatsu, Japan) according to the manufacturer’s instructions. For each cDNA synthesis, a quantity of 1 µg RNA was added to a volume of 4 µl of 5x PrimeScript RT Master Mix (Takara Bio) and stocked up to 20 µl with RNAse free ddH2O (Takara Bio). The thermocycler T100 (Bio-Rad Laboratories, Hercules, California) was set to a reverse transcription step of 15 min at 37°C, a 5 s inactivation step at 85°C and holding temperature at 4°C, according to the manufacturer’s protocol. The cDNA samples were immediately stored at -20°C.
RT-qPCR protocol and assay validation
Adequate reference gene stability of cdc42 was validated in adherence to the minimum information for publication of quantitative real-time PCR experiments (MIQE) guidelines by means of the Genorm algorithm (; ). All PCR primers contain an exon-exon junction and have an amplicon length of 70–133 nucleotides (Supplemental Table S2). Oligonucleotides were obtained from Eurofins MWG Operon LLC (Eurofins Scientific SE, Luxemburg) and primer efficiency (Ep) was determined by means of a 6x log10 serial dilution of a pooled standard cDNA solution of untreated C. elegans. In brief, reference and target genes were amplified and a standard curve was created by means of linear regression analysis in PRISM 8 software (GraphPad Software, San Diego, California). Coefficient of determination (r2) was calculated from the respective linear regression and Ep was derived from the slope: Ep = 10−1/slope -1. Acceptable Ep and r2 thresholds within the linear dynamic range (LDR) were predefined at 90–110% and r2 > 0.97, respectively. The assay specificity for each gene of interest was evaluated by melt curve analysis, with a singular peak signifying primer specificity (Supplementary Figures S1, S2). Bias from contaminating genomic DNA or primer dimers was assessed with a no-template control (NTC) for each sample and a no-reverse-transcriptase control (NRT) containing RNA instead of cDNA.
For qPCR amplification, a CFX96 Touch Real-Time PCR cycler (Bio-Rad) was used with 96-well Hard-Shell PCR plates (Bio-Rad). 20 µl mastermix, consisting of 2x iTaq Universal SYBR® Green Supermix (10 μl, Bio-Rad), the respective cDNA solution (1 µl), the respective primer pair (10 pmol/0.3 µl per primer) and nuclease-free H2O (8.4 µl, Fresenius Kabi, Bad Homburg, Germany) was administered per well. The plates were covered with Nunc sealing tape (Nalge Nunc International, Rochester, New York). Amplification of three biological replicates was carried out in technical triplicates for each gene of interest and every experimental condition. A C1000 Touch Thermal Cycler in conjunction with the CFX96 Real-Time System (Bio-Rad) was used. The amplification protocol (40 cycles, initial denaturation at 95°C/3 min, denaturation at 95°C/10 s, annealing at the respective Ta/30 s, extension at 70°C/10 s) was followed by a consecutive melt curve analysis (65°C–95°C in 0.5°C increment/5 s each).
In silico analysis and statistics
Blast analysis was performed using NCBI (; ) as well as ClustalO (; ). Statistical analysis was performed using GraphPad Prism version 7.00 for Windows (GraphPad Software, La Jolla, USA). For the determination of the optimal reaction temperature, we plotted the highest initial reaction rates against the temperature and presented as mean. For the determination of the KM, the initial reaction rates were plotted against the substrate concentration and fitted to the Michaelis-Menten equation using non-linear curve fitting of GraphPad Prism Software (GraphPad Software 8.0, San Diego, CA, USA). We calculated KM-values and its 95% confidence interval.
Based on the experimentally determined IC50 values, we calculated the inhibition constant (Ki). The relationship is described by Cheng-Prusoff (). FTase acts via ordered sequential mechanism with FPP binding first (). The following equation is applicable for Manumycin A competing with the first substrate in a bisubstrate enzyme mechanism:
The KM, KI and IC50 values are presented as the mean with 95% confidence interval. Analysis of RT-qPCR data was performed by the common base method (). In brief, Cq values were corrected for amplification efficiency following the formula CqE = log10 (Ep)*Cq and ΔCqE values were obtained by subtracting target gene values (CqEGOI) from the reference gene values (CqEREF). Next, ΔΔCqE was calculated by means of ANOVA and Tukey’s multiple comparisons test. Mean differences and 95% CI were transformed back (10−ΔΔCqE) to obtain the relative fold change. Mean differences resulting in p-values <0.05 were considered significant.
Results
Protein alignment of human and C. elegans FTase shows high conservation
To elucidate the comparability of C. elegans FTase with human FTase, we first evaluated the percentage identity of human and C. elegans FTase subunits. Therefore, we performed sequence alignments with ClustalO. The α-subunit has a query coverage of 79% with 40% identity and the β-subunit of 86% with 45.6% identity. This illustrates the high conservation of FTase throughout eukaryotes (Supplementary Figures S3, S4).
ceFTase has a high temperature optimum and comparable KMs to hFTase
Since there are no published functional parameters for the FTase from C. elegans, we determined the optimal assay temperature for the enzyme assay. hFTase has an optimal temperature of 30°C (), but due to the optimal growing temperature of C. elegans at 20°C, we performed the assay at temperatures of 15 and 20°C. Interestingly, the activity was higher at 20°C so we performed the experiments with a wider temperature range (7, 15, 20, 25, 30 and 40°C). To our surprise, the highest activity could be measured at 40°C (Figures 1A,B). Heating up the assay to 45 and 50°C led to enzyme degradation (Figure 1C) and hardly any activity was measured. Having the optimal reaction temperature, we determined the KMs for FPP (1.3 μM) and Dansyl-GCVLS (1.7 μM) (Figure 2).
FIGURE 1
FIGURE 2
Manumycin A does not totally inhibit LNCaP, HEK293 and PC3 viability
To analyze the inhibitory effect of Manumycin A (Figure 3A) in cell culture, cell viability of different cell lines (LNCaP, HEK293 and PC3) was tested after incubation with increasing amounts of Manumycin A for 48 h. The determined IC50s are 8.79 µM (LNCaP), 6.60 µM (HEK293) and 11.00 µM (PC3) (Figures 3B–D). Our results are in line with the findings of Li et al. showing a reduction of cell viability at a concentration of 30 µM Manumycin A down to 20% (). Doubling the concentration of Manumycin A does not lead to a further decrease of viability.
FIGURE 3
No inhibition of C. elegans endogenous FTase after treatment with Manumycin A
To evaluate whether Manumycin A treatment leads to an inhibition of the endogenous ceFTase, we exposed the worms to 63 nM Manumycin A, the concentration, where Bar and Gruenbaum first identified a phenotype after treatment that was not toxic (). The initial flow rates of ceFTase in the whole cell lysate from untreated worms, DMSO-treated worms (0.55%) and Manumycin A treated worms (63 nM) were akin to each other (untreated: 7.154 (95% CI 6.47-7.83), DMSO: 7.86 (95% CI 7.21-8.51) and Manumycin A: 8.11 (95% CI 7.40-8.82)) (Supplementary Figure S5).
High IC50 values for Manumycin A in FTase cell-free assay
To investigate whether the described effects of Manumycin A in cell culture assays could depend on the inhibition of FTase or are related to other mechanisms in the cells, we determined the IC50 for human and C. elegans IMAC purified FTase using increasing Manumycin A concentrations at the optimal assay temperature for human (30°C) and ceFTase (40°C). For hFTase, we determined an IC50 of 58.03 μM and a Ki of 4.40 μM (CI 3.68–5.25 µM)—for ceFTase the IC50 value is 45.96 μM and the Ki 3.16 μM (CI 2.43–4.31 µM) (Figures 3E,F). The high IC50 values indicate that a quite high concentration of Manumycin A is needed to inhibit the FTase. Around 8.8 µM Manumycin A is needed to have a 50% reduction in cell viability. This is five times less compared to the cell-free assay. Interestingly, when measuring the IC50 of ceFTase at a much lower temperature (25°C), we got an even higher value with 117.3 µM (CI 69.3–191.3 µM) (Supplementary Figure S6).
Manumycin A but not the TrxR inhibitor D-9 up-regulates fnta and fntb gene transcription in a dose dependent manner in C. elegans
To elucidate whether expression of the farnesyltransferase genes fnta and fntb is influenced by the addition of Manumycin A, RT-qPCR was performed. The worms were exposed to either 3, 9,25, 50 or 100 μM of Manumycin A or DMSO. The data show that relative gene expression of farnesyltransferase genes fnta and fntb was upregulated in response to the varying doses of Manumycin A (Figure 4). Moreover, fntb transcription was up-regulated in a dose-dependent manner. After incubation, the expression of fnta increased 1.3-fold (3 µM) to 4.7-fold (25 µM) in response to Manumycin A. However, the mean fnta expression in DMSO treated and Manumycin A treated groups were not significantly different. fntb expression increased dose-dependently from initial downregulation by 0.15-fold up to a 4.2-fold upregulation. The mean differences between the worms treated with 100 µM Manumycin A and the DMSO treated worms were statistically not significant either (Figure 4). These results indicate a dose dependent influence on C. elegans farnesyltransferase expression but only at very high Manumycin A concentrations, supporting the findings from the cell-free assay. In response to treatment with various concentrations of the TrxR inhibitor D-9, neither fnta nor fntb transcription exhibited considerable upregulation. On the contrary, fnta was significantly down-regulated by D-9 in concentrations of 10, 30 and 300 nM (Supplementary Figure S7).
FIGURE 4
Manumycin A induces C. elegans apoptosis pathways via cell-death protein 4
In addition to the farnesyltransferase genes fnta and fntb, we investigated two genes that are involved in C. elegans apoptosis, namely cell death protein 3 and 4 (CED-3 and CED-4). CED-3 is an analogous to the human apoptotic protease activating factor-1 (APAF-1) and CED-4 to Caspase-9. In response to Manumycin A, the relative gene expression of ced4 was significantly up-regulated in a dose-dependent manner from 3 to 25 µM (27-fold–123-fold), followed by a 14.7-fold and 10.9-fold up regulation for 50 μM and 100 μM Manumycin A, respectively (Figure 4). On the other hand, ced3, a Caspase analogue, was not or inversely regulated by Manumycin A. Similarly, the effect of D-9 led to a significant increase of ced4 transcription, ranging from 12-fold to 16-fold, accompanied by a non-significant down-regulation of ced3 (Supplementary Figure S6). From the data obtained, one could assume that this effect relies on a CED-3 independent apoptotic event in response to both, Manumycin A and D-9 (; ).
Discussion
This is the first time that kinetic data for ceFTase are determined. Surprisingly, ceFTase shows its maximal activity at 40°C, even though the worm´s cell cycle, cell division and reproduction are negatively affected by temperatures above 25°C (). Although C. elegans has thermosensory neurons, all tests with it have only been performed up to 25°C. In this study the authors proposed that the thermosensory system helps C. elegans to withstand higher temperatures (). However, a study by Gómez-Orte et al. could show, that C. elegans shows not only a changed transcriptome by temperature alterations but also by the kind of applied diet (). Worms fed on E. coli show no regulation of FTase independent of the temperature. When fed on Bacillus subtilis, a transcriptional upshift of fnta could be observed at 15°C (). The high temperature optimum for ceFTase is not a uniqueness. This could also be observed for malat dehydrogenases 1 and 2 of C. elegans having their maximal activity at 40°C and 35°C, respectively ().
The KM values for FTase that can be found in literature differ quite widely depending on the organism and the assay used. The value for Dansyl-GCVLS from Rattus norvegicus (0.7 μM) is comparable with 1.7 μΜ for C. elegans (). The KM-values for FPP found in the literature range from 0.2 nM in Plasmodium falciparum () to 46 μΜ for Rattus norvegicus ().
So far, no kinetic data on the inhibitory property of Manumycin A as an FTI existed. With our data, we can demonstrate that Manumycin A is capable of inhibiting human or C. elegans FTase in a cell-free assay, but not in pharmacologically relevant concentrations. The IC50 and Ki values are relatively high (IC50 58.03 μM/Ki 4.40 µM for human and IC50 45.96 μM/Ki 3.16 µM for C. elegans FTase). Lonafarnib and tipifarnib, the most prominent FTIs, show 24000 times lower IC50 values of 1.9 nM and 0.86 nM, respectively (; ). These FTIs are not competitive inhibitors against FPP like Manumycin A, but against the CAAX substrate. Other known FPP analogues like α-hydroxyfarnesylpyrophosphate show 850-fold lesser values (Ki of 5.2 nM) in bovine brain ().
Since many studies prove Manumycin A to induce apoptosis, we investigated the effect in vitro using LNCaP, HEK293 and PC3 cells. Li et al. showed that the cell viability of LNCaP cells is reduced to 20% after treatment with 30 μM Manumycin A (). Our findings are in line with that and with that of other studies showing IC50 values in different cell lines between 4.3 and 50 µM Manumycin A (; ; ; ; ). Like Li et al., we were not able to induce a complete inhibition of cell growth, even when using concentrations of 100 μM Manumycin A. Importantly, the concentrations to reduce cell viability to 50% were far below those required to inhibit FTase in our cell-free assay, indicating that Manumycin A results in apoptosis independent of FTase inhibition. Furthermore, we did not observe an inhibition of endogenous FTase in the worms’ whole cell lysate after Manumycin A treatment.
On the RNA level, the cell-free determined IC50 concentration (50 µM Manumycin A) leads to no relevant regulation of fnta and fntb. Only after doubling the Manumycin A concentration, we see a slide upshift of the RNA expression of both genes. However, we can only see a dose-dependent increase of the fntb expression, but not for fnta, leading to the question, whether this is a direct effect of the Manumycin A treatment or just a side effect. It could be shown, that treatment with 10 µM of the highly potent FTI-276 reduces the FTase activity but upregulates the expression von FNTA and FNTB in human rheumatoid synovial fibroblasts (). Manumycin A leads to the activation of apoptotic genes, seen by a 14.7-fold upregulation of ced4. To our knowledge, ced4 is not directly connected to treatment with Manumycin A. Therefore, we expanded our literature research on effects of Manumycin A independent of FTase inhibition.
Various studies have highlighted single aspects of Manumycin A influencing apoptotic pathways, like TrxR-1, NF-κB, pAKT, PI3K, the Bcl-2 family as well as the proteasome, to name just a few (; ; ; ; ). In contrast, most studies using Manumycin A as an FTI refer to Hara et al. () who described the reagent to be a highly potent and specific FTI in yeast, but some studies doubt the role of Manumycin A as an FTI ().
To our knowledge, this is the first paper that brings prominent effects of Manumycin A in a context, explaining most of the effects and showing that its role as FTI is neglectable for apoptosis in cancer cells (Figure 5). It was shown that Manumycin A inhibits thioredoxin reductase 1 (TrxR1) with an IC50 of 272 nM, resulting both in accumulation of reactive oxygen species (ROS) and in activation of the xenobiotic apoptotic pathway (; ). The latter comprises apoptosis regulating kinase 1 (ASK1), p38 mitogen activated protein kinase (MAPK)-pathway as well as c-Jun NH2-terminal Kinase (c-JNK) () and it has been shown that this xenobiotic apoptotic pathway is upregulated after treatment with 54 μM Manumycin A (; ; ). The accumulation of ROS on the other hand leads to inhibition of the NF-κB pathway and thus to reduced expression of anti-apoptotic proteins like Bcl-XL or Baf (). Other groups showed that BclXL is downregulated by Manumycin A () resulting in less anti-apoptotic proteins. This downregulation of BclXL and upregulation of Bax was also observed by Kim et al. after the addition of 2.5–10 µΜ Manumycin A (). In line with that, Bernier et al. postulated, that 10 μM Manumycin A inhibits selectively IKKβ, an upstream regulator of NF-κB (). Irrespective of NF-κB inhibition, accumulation of ROS in response to Manumycin A was also observed by Sears et al. () leading to the activation of Cas-9 and Cas-3, triggering apoptosis as well. In line with that, the effect of Manumycin A on ROS elevation and H2O2 loss was also shown by Carey et al. after an administration of 4 μM Manumycin A (). An upregulation of Cas-3 after Manumycin A treatment could also be observed in other studies ().
FIGURE 5
Furthermore, another important effect of Manumycin A is the induction of chaperone expression as well as the induction of endoplasmic reticulum stress (ER stress). It was shown that Manumycin A increases the amount of polyubiquitinated proteins and inhibits the proteasome (). All this leads to the expression of the proapoptotic protein NOXA that is normally degraded by the proteasome. Due to the higher amount of NOXA, Caspase-9 is activated, leading subsequently to apoptosis (; ).
Interestingly these data from human cell lines are in line with our experimental results regarding the regulation of the C. elegans cell death proteins ced-3 and ced-4 (Figure 5, inner box). ced-3 is the corresponding gene to the human cas-9 and ced-4 to apaf, respectively () (Figure 5). In both organisms, APAF/CED-4, induce CAS-9/CED-3. In contrast to the human system, it was shown, that elevated mitochondrial ROS levels in C. elegans result in longevity by activating the BH3-only protein and therefore CED-9 and CED-4 (). Accordingly, Manumycin A treatment prolongs C. elegans motility (). The expression of ced-4 was 15-fold upregulated after Manumycin A treatment. Yee at al. also proposed that CED-3 is not as important as CED-4 for this reaction reflected by our findings that there is no expressional regulation of ced-3 after Manumycin A treatment.
Summarizing all these data, we hypothesize that Manumycin A is neither a highly potent nor a specific FTI. We rather propose that it has multi-plane effects on the highly cross-linked network of apoptotic pathways. From our point of view, the inhibition of TrxR1 is the main key player inducing apoptosis via different pathways that influence each other. On the one hand, there is an increase in ROS and a decrease in H2O2, highly having impact. On the other hand, ASK1 cannot be inhibited which induces apoptosis. All the mentioned effects lead to the activation of caspases and subsequently to apoptosis. The exact mechanisms how all these rack-wheels interact need to be elucidated in further studies, taking our findings on the role of Manumycin A as new starting point.
Statements
Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.
Author contributions
HB: project conceptualization. AH, LW, and PA: methodology. AH, DJ, LW, PA, MK, and DD: formal analysis and investigation. AH, PA, and HB: manuscript writing. HB, DJ, AH, LW, and MK: manuscript review and editing. PA, DD, and AH: data visualization. HB: supervision. All authors contributed to the article and approved the submitted version.
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Publisher’s note
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Supplementary material
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fchem.2022.967947/full#supplementary-material
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Summary
Keywords
farnesyltransferase, Manumycin A, apoptosis, prenylation, Caenorhabditis elegans
Citation
Hagemann A, Altrogge PK, Kehrenberg MCA, Diehl D, Jung D, Weber L and Bachmann HS (2022) Analyzing the postulated inhibitory effect of Manumycin A on farnesyltransferase. Front. Chem. 10:967947. doi: 10.3389/fchem.2022.967947
Received
13 June 2022
Accepted
24 November 2022
Published
06 December 2022
Volume
10 - 2022
Edited by
Rosana I. Misico, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina
Reviewed by
Parmanand Malvi, University of Alabama at Birmingham, United States
Ashutosh Kumar Pandey, The State University of New Jersey, United States
Shizhong Dai, Stanford University, United States
Updates
Copyright
© 2022 Hagemann, Altrogge, Kehrenberg, Diehl, Jung, Weber and Bachmann.
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Hagen Sjard Bachmann, hagen.bachmann@uni-wh.de
† These authors have contributed equally to this work and share first authorship
This article was submitted to Chemical Biology, a section of the journal Frontiers in Chemistry
Disclaimer
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.