Hagen Sjard Bachmann

Farnesyltransferase inhibitors (FTIs) like tipifarnib and lonafarnib, originally developed as anticancer agents, show antibacterial activity against Gram-positive pathogens through an unknown mechanism of action. Here, we investigated the proteomic response of Bacillus subtilis 168 (Gram-positive) and an efflux-deficient Escherichia coli Δ tolC mutant (Gram-negative) to sublethal concentrations of FTIs. Concentrations resulting in approximately 50% growth inhibition were applied, followed by two complementary proteomic approaches: gel-free quantitative LC-HDMS ^E (both organisms) and 2D-PAGE with [ ³⁵ S]-methionine pulse labelling of newly synthesized proteins ( B. subtilis ). Marker proteins from the B. subtilis 2D-PAGE approach were used for comparative proteomic response profiling against a reference library of 96 antibacterial agents. Bacillus subtilis was more susceptible to both FTIs than E. coli Δ tolC , with tipifarnib being more potent. LC-HDMS ^E analysis revealed a pronounced cell envelope stress signature in B. subtilis , including regulation of YocH, YabE, and YjhA, along with several σ ^B , σ ^W , and σ ^M -regulated stress proteins. In the 2D-PAGE analysis, reflecting acute cellular responses by monitoring newly synthesized proteins, both FTIs induced proteins such as ClpC, PspA, and LiaH. Comparative proteomic response analysis in B. subtilis showed that both FTIs cluster with membrane-active antibiotics, including xanthoangelol, MP196, gramicidin S, MP66, and nisin, which binds the cell wall precursor lipid II and forms pores in the bacterial membrane. Similarities were also observed with pseudopterosin G and pseudopteroxazole, which target the bacterial cell envelope by integration into the membrane and interfering with membrane-bound steps of cell wall biosynthesis. Yet, the similarity of the proteomic profiles does not indicate a perfect match or an identical molecular mode of action within the reference library. In E. coli Δ tolC , the proteomic response was less pronounced, but for treatment with tipifarnib, regulated proteins of the category “membrane-bounded periplasmic space” were enriched. Proteomic profiling, combined with functional validation via membrane integrity and membrane potential assays, indicates that FTIs compromise the bacterial membrane and activate stress and repair pathways. The FTIs may also indirectly affect cell wall biosynthesis. The lack of a perfect match with any of the 96 compounds in the proteomic reference library highlights FTIs as promising lead structures for the development of novel antibiotics.

Background: Protein prenylation is crucial for the function of hundreds of proteins. Aberrant protein prenylation can be caused by the aberrant expression of prenyltransferases (PTases), which has been reported for multiple cancer entities. The reasons for aberrant PTase expression in cancer have not yet been investigated. Methods: We analyzed CpG methylation within promoter-associated CpG islands of the PTase genes FNTA, FNTB, PGGT1B, and RABGGTA via bisulfite conversion and pyrosequencing to assess its role in PTase expression and gain deeper insight into the regulation of protein prenylation in cancer. We used DNA from three benign controls (whole blood samples, peripheral blood mononuclear cells, and HEK293) and 19 human cancer cell lines from various origins to assess DNA methylation within PTase gene promoter-associated CpG islands. For a subset of these cell lines, we measured mRNA expression via qPCR and correlated it with DNA methylation. Results: Methylation across all PTase genes ranged from 1.9 ± 0.9% to 11.4 ± 4.0% (mean methylation ± standard deviation) in benign cells, and 2.3 ± 1.0% to 16.0 ± 5.4% in cancer cells. DNA methylation and mRNA expression of PGGT1B correlated inversely (PCC = −0.75; p = 0.005). Conclusions: We saw no general differences between benign and malignant cells, but observed significant differences between non-malignant controls and multiple individual cancer cell lines regarding the methylation of PTase genes. This was prominently seen in PGGT1B in Caki-1 cells, raising the possibility that DNA methylation is involved in the dysregulation of PTase expression in cancer.

Background: Farnesyltransferase inhibitors (FTIs) were developed for preventing the prenylation of aberrant Ras in human cancer. Furthermore, prenylation is involved in multiple biological processes and plays a putative role in signalling pathways such as the Ras-mitogen-activated-protein kinase (MAPK) pathway. Matrix-vesicle-mediated mineralization (MVM) is the first step in the development of eukaryotic mineralized tissue. In this study, we evaluated the effect of FTIs on MVM in the osteosarcoma cell line SaOS-2 and elucidated the role of farnesylation in this process. Methods: SaOS-2 cells were treated with the FTIs Lonafarnib and Tipifarnib. Mineralization was assessed using Alizarin Red S staining. Enzyme assays were conducted to measure alkaline phosphatase (ALP) activity. Western blot analysis and Oxford Nanopore sequencing were performed to evaluate the expression of ALP, collagen type I (COL1A1), and Runt-related transcription factor 2 (RUNX2). Results: Inhibition of farnesylation by FTIs resulted in decreased mineralization, as evidenced by reduced Alizarin Red S staining. Additionally, RUNX2 activity was diminished, leading to a reduction in MVM and decreased expression of ALP and COL1A1. Conclusion: Our findings demonstrate that FTIs Lonafarnib and Tipifarnib impair MVM, highlighting the essential role of farnesylation in biomineralization.

The emergence of antibiotic resistance continues to pose a significant global challenge. Drug repurposing, wherein existing therapeutics are evaluated for new applications, offers a promising strategy to address this issue. Farnesyltransferase inhibitors (FTIs), initially developed for cancer therapy, have demonstrated antimicrobial activity against several gram-positive bacteria. This study investigates their activity in combination with colistin against gram-positive and gram-negative bacteria. We focus on key ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens while incorporating additional bacterial strains to provide a comprehensive understanding of differential responses and potential dose-dependent synergistic effects. Antimicrobial susceptibility was assessed using broth microdilution, while synergy was evaluated through checkerboard, time-kill, and growth kinetics assays. When combined with sub-inhibitory colistin, FTIs inhibited gram-negative bacterial growth. Tipifarnib exhibited more potent antimicrobial activity against gram-negative strains than lonafarnib. Peptidomimetic FTIs, B581 and FTI-277, inhibited gram-negative bacteria in combination with colistin but had no effect on the gram-positive strains tested. In contrast, alpha-hydroxy farnesyl phosphonic acid, an FPP analog, and bempedoic acid, targeting the mevalonate pathway, showed no antibacterial activity. In addition to their known inhibition of gram-positive bacteria, FTIs exhibited efficacy against gram-negative bacteria, including colistin-resistant Enterobacter cloacae subsp. cloacae, when combined with sub-inhibitory colistin. This might be due to a mechanism distinct from their eukaryotic targets, potentially involving the disruption of multiple biosynthetic pathways. Future studies will focus on elucidating these mechanisms of FTIs and exploring the therapeutic potential of FTI/colistin combinations against ESKAPE and other multidrug-resistant pathogens.

Background: Protein prenylation is crucial for the function of hundreds of proteins. Aberrant protein prenylation can be caused by aberrant expression of prenyltransferases (PTases), which has been reported for multiple cancer entities. The reasons for aberrant PTase expression in cancer have not yet been investigated. Methods: We analyzed CpG methylation within promoter-associated CpG islands of PTase genes FNTA, FNTB, PGGT1B and RABGGTA via bisulfite conversion and pyrosequencing to assess its role in PTase expression and gain deeper insight into the regulation of protein prenylation in cancer. We used DNA from three benign controls (whole blood samples, peripheral blood mononuclear cells, HEK293) and 19 human cancer cell lines from various origins to assess DNA methylation within PTase gene promoter-associated CpG islands. For a subset of these cell lines, we measured mRNA expression via qPCR and correlated it with DNA methylation. Results: Methylation across all PTase genes ranged from 1.9 % ± 0.9 to 11.4 % ± 4.0 (mean methylation ± standard deviation) in benign cells, and 2.3 % ± 1.0 to 16.0 % ± 5.4 in cancer cells. DNA methylation and mRNA expression of PGGT1B correlated inversely (PCC = −0.75; p = 0.005). Conclusions: We saw no general differences between benign and malignant cells, but observed significant differences between non-malignant controls and multiple individual cancer cell lines regarding methylation of PTase genes, most prominently of FNTA and PGGT1B in Caki-1 and Kelly cells, raising the possibility that DNA methylation is involved in the dysregulation of PTase expression in cancer.

Tuberous sclerosis complex (TSC) is a rare inherited disease with the potential to affect virtually every organ system. Clinical presentation is age- and partly sex-dependent and varies broadly with respect to disease manifestations including treatment-refractory epilepsy, intellectual disability and TSC-associated neuropsychiatric disorders, chronic kidney disease or progressive lung function decline. Given the complexity of this disease, multidisciplinary care in specialized TSC centres is recommended. We aimed to elucidate the state of knowledge of patients/caregivers and physicians on individual disease manifestations. We further examined whether the association to a TSC centre has an impact on the comprehensive consideration of potential disease manifestations. Therefore, a survey was performed in a cohort of German TSC patients and their physicians. Complete information was available for 94 patients with a median age of 18 years [range 1–55] and a sex distribution of 53.2% (male): 48.8% (female). Using almost identical questionnaires for patients/caregivers and their respective physician, there was a good correlation for disease assessments associated with relevant morbidity and mortality like epilepsy, renal angiomyolipoma, cardiac rhabdomyomas or intellectual disability. Correlation was moderate for several neuropsychiatric disorders and only poor for hypomelanotic macules, dental pits or retinal achromic patches. Estimation of overall disease severity using a numeric rating scale correlated highly significantly (Pearson correlation coefficient = 0.767; p < 0.001) between patients/caregivers and physicians. In general, physicians more likely quoted items as ‘unknown’ than patients (822 answers vs. 435 answers in the respective groups). Questionnaires completed by physicians who were associated with a specialized TSC centre declared a significantly lower proportion of items as unknown (mean 8.7% vs. 20.5%; p < 0.001). These findings indicate that patients treated by specialized TSC centres seem to obtain a more comprehensive surveillance. Furthermore, it shows that there were reasonable surveillance strategies in general and sufficient patient/caregiver interaction and education in the examined cohort. However, for the most prominent disease characteristics there was a good awareness within both the patients/caregivers and the physicians group.

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 K_i 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 K_M 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 (K_{i human} 4.15 μM; K_{iC. 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.

The CAAX-prenyltransferases farnesyltransferase (FTase) and geranylgeranyltransferase I (GGTase I) are heterodimers with a common α- (FTα) and unique β-subunits. Recently, α-subunits of species (e.g., human) that harbour an N-terminal proline-rich region (PRR) showed different dimerization behaviours than α-subunits without PRR (e.g., yeast). However, the specific function of the PRR has not been elucidated so far. To determine whether the PRR is a conserved motif throughout eukaryotes, we performed phylogenetics. Elucidating the impact of the PRR on enzyme properties, we cloned human as well as rat PRR deficient FTα, expressed them heterologously and compared protein–protein interaction by pull-down as well as crosslinking experiments. Substrate binding, enzyme activity and sensitivity towards common FTase inhibitors of full length and PRR-deletion α-subunits and their physiological partners was determined by continuous fluorescence assays. The PRR is highly conserved in mammals, with an exception for marsupials harbouring a poly-alanine region instead. The PRR shows similarities to canonical SH3-binding domains and to profilin-binding domains. Independent of the PRR, the α-subunits were able to dimerize with the different physiological β-subunits in in vitro as well as in yeast two-hybrid experiments. FTase and GGTase I with truncated FTα were active. The K_M values for both substrates are in the single-digit µM range and show no significant differences between enzymes with full length and PRR deficient α-subunits within the species. Our data demonstrate that an N-terminal PRR of FTα is highly conserved in mammals. We could show that the activity and inhibitability is not influenced by the truncation of the N-terminal region. Nevertheless, this region shows common binding motifs for other proteins involved in cell-signalling, trafficking and phosphorylation, suggesting that this PRR might have other or additional functions in mammals. Our results provide new starting points due to the relevant but only partly understood role of FTα in eukaryotic FTase and GGTase I. Video Abstract