TRIM28 Selective Nanobody Reduces Glioblastoma Stem Cell Invasion.

Glioblastoma (GB), is the most common and aggressive malignant primary brain tumour in adults. Intra- and inter-tumour heterogeneity, infiltrative GB cell invasion and presence of therapy-resistant GB stem cells (GSCs) represent major obstacles to favourable prognosis and poor therapy response. Identifying the biomarkers of the most aggressive tumour cells and their more efficient targeting strategies are; therefore, crucial. Recently, transcription factor TRIM28 has been identified as a GB biomarker and, in this study, we have shown high expression of TRIM28 in GB and in low grade gliomas as well as higher expression in GSCs vs. differentiated GB cells, although in both cases not significant. We demonstrated significant in vitro inhibition of GB cells and GSCs invasiveness and spread in zebrafish brains in vivo by anti-TRIM28 selective nanobody NB237. TRIM28 was also enriched in GB (tumour) core and associated with the expression of stem cell genes, but was not prognostic for overall survival. However, based on the above results, we conclude that TRIM28 nanobody NB237 offers a new opportunity as a GB therapeutic tool.

Analysis of miR-9-5p, miR-124-3p, miR-21-5p, miR-138-5p, and miR-1-3p in Glioblastoma Cell Lines and Extracellular Vesicles.

Glioblastoma (GBM), the most common primary brain tumor, is a complex and extremely aggressive disease. Despite recent advances in molecular biology, there is a lack of biomarkers, which would improve GBM's diagnosis, prognosis, and therapy. Here, we analyzed by qPCR the expression levels of a set of miRNAs in GBM and lower-grade glioma human tissue samples and performed a survival analysis in silico. We then determined the expression of same miRNAs and their selected target mRNAs in small extracellular vesicles (sEVs) of GBM cell lines. We showed that the expression of miR-21-5p was significantly increased in GBM tissue compared to lower-grade glioma and reference brain tissue, while miR-124-3p and miR-138-5p were overexpressed in reference brain tissue compared to GBM. We also demonstrated that miR-9-5p and miR-124-3p were overexpressed in the sEVs of GBM stem cell lines (NCH421k or NCH644, respectively) compared to the sEVs of all other GBM cell lines and astrocytes. VIM mRNA, a target of miR-124-3p and miR-138-5p, was overexpressed in the sEVs of U251 and U87 GBM cell lines compared to the sEVs of GBM stem cell line and also astrocytes. Our results suggest VIM mRNA, miR-9-5p miRNA, and miR-124-3p miRNA could serve as biomarkers of the sEVs of GBM cells.

Characterization and risk association of polymorphisms in Aurora kinases A, B and C with genetic susceptibility to gastric cancer development.

BACKGROUND: Single nucleotide polymorphisms (SNPs) in genes encoding mitotic kinases could influence development and progression of gastric cancer (GC). METHODS: Case-control study of nine SNPs in mitotic genes was conducted using qPCR. The study included 116 GC patients and 203 controls. In silico analysis was performed to evaluate the effects of polymorphisms on transcription factors binding sites. RESULTS: The AURKA rs1047972 genotypes (CT vs. CC: OR, 1.96; 95% CI, 1.05-3.65; p = 0.033; CC + TT vs. CT: OR, 1.94; 95% CI, 1.04-3.60; p = 0.036) and rs911160 (CC vs. GG: OR, 5.56; 95% CI, 1.24-24.81; p = 0.025; GG + CG vs. CC: OR, 5.26; 95% CI, 1.19-23.22; p = 0.028), were associated with increased GC risk, whereas certain rs8173 genotypes (CG vs. CC: OR, 0.60; 95% CI, 0.36-0.99; p = 0.049; GG vs. CC: OR, 0.38; 95% CI, 0.18-0.79; p = 0.010; CC + CG vs. GG: OR, 0.49; 95% CI, 0.25-0.98; p = 0.043) were protective. Association with increased GC risk was demonstrated for AURKB rs2241909 (GG + AG vs. AA: OR, 1.61; 95% CI, 1.01-2.56; p = 0.041) and rs2289590 (AC vs. AA: OR, 2.41; 95% CI, 1.47-3.98; p = 0.001; CC vs. AA: OR, 6.77; 95% CI, 2.24-20.47; p = 0.001; AA+AC vs. CC: OR, 4.23; 95% CI, 1.44-12.40; p = 0.009). Furthermore, AURKC rs11084490 (GG + CG vs. CC: OR, 1.71; 95% CI, 1.04-2.81; p = 0.033) was associated with increased GC risk. A combined analysis of five SNPs, associated with an increased GC risk, detected polymorphism profiles where all the combinations contribute to the higher GC risk, with an OR increased 1.51-fold for the rs1047972(CT)/rs11084490(CG + GG) to 2.29-fold for the rs1047972(CT)/rs911160(CC) combinations. In silico analysis for rs911160 and rs2289590 demonstrated that different transcription factors preferentially bind to polymorphic sites, indicating that AURKA and AURKB could be regulated differently depending on the presence of particular allele. CONCLUSIONS: Our results revealed that AURKA (rs1047972 and rs911160), AURKB (rs2241909 and rs2289590) and AURKC (rs11084490) are associated with a higher risk of GC susceptibility. Our findings also showed that the combined effect of these SNPs may influence GC risk, thus indicating the significance of assessing multiple polymorphisms, jointly. The study was conducted on a less numerous but ethnically homogeneous Bosnian population, therefore further investigations in larger and multiethnic groups and the assessment of functional impact of the results are needed to strengthen the findings.

Meta-Analysis and Experimental Validation Identified FREM2 and SPRY1 as New Glioblastoma Marker Candidates.

Glioblastoma (GB) is the most aggressive brain malignancy. Although some potential glioblastoma biomarkers have already been identified, there is a lack of cell membrane-bound biomarkers capable of distinguishing brain tissue from glioblastoma and/or glioblastoma stem cells (GSC), which are responsible for the rapid post-operative tumor reoccurrence. In order to find new GB/GSC marker candidates that would be cell surface proteins (CSP), we have performed meta-analysis of genome-scale mRNA expression data from three data repositories (GEO, ArrayExpress and GLIOMASdb). The search yielded ten appropriate datasets, and three (GSE4290/GDS1962, GSE23806/GDS3885, and GLIOMASdb) were used for selection of new GB/GSC marker candidates, while the other seven (GSE4412/GDS1975, GSE4412/GDS1976, E-GEOD-52009, E-GEOD-68848, E-GEOD-16011, E-GEOD-4536, and E-GEOD-74571) were used for bioinformatic validation. The selection identified four new CSP-encoding candidate genes—CD276, FREM2, SPRY1, and SLC47A1—and the bioinformatic validation confirmed these findings. A review of the literature revealed that CD276 is not a novel candidate, while SLC47A1 had lower validation test scores than the other new candidates and was therefore not considered for experimental validation. This validation revealed that the expression of FREM2—but not SPRY1—is higher in glioblastoma cell lines when compared to non-malignant astrocytes. In addition, FREM2 gene and protein expression levels are higher in GB stem-like cell lines than in conventional glioblastoma cell lines. FREM2 is thus proposed as a novel GB biomarker and a putative biomarker of glioblastoma stem cells. Both FREM2 and SPRY1 are expressed on the surface of the GB cells, while SPRY1 alone was found overexpressed in the cytosol of non-malignant astrocytes.

Erythropoietin and Its Angiogenic Activity.

Erythropoietin (EPO) is the main hematopoietic hormone acting on progenitor red blood cells via stimulation of cell growth, differentiation, and anti-apoptosis. However, its receptor (EPOR) is also expressed in various non-hematopoietic tissues, including endothelium. EPO is a pleiotropic growth factor that exhibits growth stimulation and cell/tissue protection on numerous cells and tissues. In this article we review the angiogenesis potential of EPO on endothelial cells in heart, brain, and leg ischemia, as well as its role in retinopathy protection and tumor promotion. Furthermore, the effect of EPO on bone marrow and adipose tissue is also discussed.

Association of the AURKA and AURKC gene polymorphisms with an increased risk of gastric cancer.

Single nucleotide polymorphisms (SNPs) in mitotic checkpoint genes can contribute to susceptibility of human cancer, including gastric cancer (GC). We aimed to investigate the effects of Aurora kinase A (AURKA), Aurora kinase B (AURKB), and Aurora kinase C (AURKC) gene polymorphisms on GC risk in Slovenian population. We genotyped four SNPs in AURKA (rs2273535 and rs1047972), AURKB (rs2241909), and AURKC (rs758099) in a total of 128 GC patients and 372 healthy controls using TaqMan allelic discrimination assays to evaluate their effects on GC risk. Our results showed that genotype frequencies between cases and controls were significantly different for rs1047972 and rs758099 (P < 0.05). Our study demonstrated that AURKA rs1047972 TT and (CC + CT) genotypes were significantly associated with an increased risk of gastric cancer. Our results additionally revealed that AURKC rs758099 TT and (CC + CT) genotypes were also associated with increased GC risk. In stratified analysis, genotypes TT and (CC + CT) of AURKA rs1047972 SNP were associated with increased risk of both, intestinal and diffuse, types of GC. In addition, AURKC rs758099 TT and (CC + CT) genotypes were positively associated with increased intestinal type GC risk, but not with an increased diffuse type GC risk. Based on these results, we can conclude that AURKA rs1047972 and AURKC rs758099 polymorphisms could affect the risk of GC development. Further larger studies are needed to confirm these findings. © 2016 IUBMB Life, 68(8):634-644, 2016.

Association between polymorphisms in segregation genes BUB1B and TTK and gastric cancer risk.

BACKGROUND: Malignant transformation of normal gastric cells is a complex and multistep process, resulting in development of heterogeneous tumours. Susceptible genetic background, accumulation of genetic changes, and environmental factors play an important role in gastric carcinogenesis. Single nucleotide polymorphisms (SNPs) in mitotic segregation genes could be responsible for inducing the slow process of accumulation of genetic changes, leading to genome instability. PATIENTS AND METHODS: We performed a case-control study of polymorphisms in mitotic kinases TTK rs151658 and BUB1B rs1031963 and rs1801376 to assess their effects on gastric cancer risk. We examined the TTK abundance in gastric cancer tissues using immunoblot analysis. RESULTS: C/G genotype of rs151658 was more frequent in patients with diffuse type of gastric cancer and G/G genotype was more common in intestinal types of gastric cancers (p = 0.049). Polymorphic genotype A/A of rs1801376 was associated with higher risk for developing diffuse type of gastric cancer in female population (p = 0.007), whereas A/A frequencies were increased in male patients with subserosa tumour cell infiltration (p = 0.009). T/T genotype of rs1031963 was associated with well differentiated tumours (p = 0.035). TT+CT genotypes of rs1031963 and GG+AG genotypes of rs1801376 were significantly associated with gastric cancer risk (dominant model; OR = 2,929, 95% CI: 1.281-6.700; p = 0.017 and dominant model; OR = 0,364, 95% CI: 0.192-0.691; p = 0.003 respectively). CONCLUSIONS: Our results suggest that polymorphisms in mitotic kinases TTK and BUB1B may contribute to gastric tumorigenesis and risk of tumour development. Further investigations on large populations and populations of different ethnicity are needed to determine their clinical utility.

Contemporary proteomic strategies for clinical epigenetic research and potential impact for the clinic.

Novel proteomic methods are revealing the intricacy of the epigenetic landscape affecting gene regulation and improving our knowledge of the pathogenesis of complex diseases. Despite the enormous amount of data regarding epigenetic modifications present in DNA and histones, deciphering their biological relevance in the context of the disease and health is currently still an ongoing process. Here, we consider the relationship between epigenetic research in tumorigenesis and the prospect of knowledge transfer to clinical use, focusing primarily on the epigenetic histone post-translational modifications, which could be used as biomarkers. We additionally focus on the use of proteomic techniques in research and evaluate their usefulness in clinical setting.

Benzoic acid derivatives with improved antifungal activity: Design, synthesis, structure-activity relationship (SAR) and CYP53 docking studies.

Previously, we identified CYP53 as a fungal-specific target of natural phenolic antifungal compounds and discovered several inhibitors with antifungal properties. In this study, we performed similarity-based virtual screening and synthesis to obtain benzoic acid-derived compounds and assessed their antifungal activity against Cochliobolus lunatus, Aspergillus niger and Pleurotus ostreatus. In addition, we generated structural models of CYP53 enzyme and used them in docking trials with 40 selected compounds. Finally, we explored CYP53-ligand interactions and identified structural elements conferring increased antifungal activity to facilitate the development of potential new antifungal agents that specifically target CYP53 enzymes of animal and plant pathogenic fungi.

Effects of Flavonoids from Food and Dietary Supplements on Glial and Glioblastoma Multiforme Cells.

Quercetin, catechins and proanthocyanidins are flavonoids that are prominently featured in foodstuffs and dietary supplements, and may possess anti-carcinogenic activity. Glioblastoma multiforme is the most dangerous form of glioma, a malignancy of the brain connective tissue. This review assesses molecular structures of these flavonoids, their importance as components of diet and dietary supplements, their bioavailability and ability to cross the blood-brain barrier, their reported beneficial health effects, and their effects on non-malignant glial as well as glioblastoma tumor cells. The reviewed flavonoids appear to protect glial cells via reduction of oxidative stress, while some also attenuate glutamate-induced excitotoxicity and reduce neuroinflammation. Most of the reviewed flavonoids inhibit proliferation of glioblastoma cells and induce their death. Moreover, some of them inhibit pro-oncogene signaling pathways and intensify the effect of conventional anti-cancer therapies. However, most of these anti-glioblastoma effects have only been observed in vitro or in animal models. Due to limited ability of the reviewed flavonoids to access the brain, their normal dietary intake is likely insufficient to produce significant anti-cancer effects in this organ, and supplementation is needed.

Two-dimensional gel electrophoresis of gastric tissue in an alkaline pH range.

2DE in combination with MS has facilitated the discovery of several proteins with altered abundance in gastric cancer. While acidic and wide pH ranges have been widely investigated, analysis in the alkaline pH range has not been specifically performed in gastric cancer to date. In the present study, we initially optimized the 2DE in alkaline pH range (pH 7-11) for gastric tissue samples. Using a modified lysis buffer, we analyzed pooled nontumor and tumor samples for proteins with altered abundance in gastric adenocarcinoma. We successfully identified 38 silver-stained spots as 24 different proteins. Four of these were chosen for investigation with immunoblotting on individual paired samples to determine whether the changes seen in 2DE represent the overall abundance of the protein or possibly only a single form. While mitochondrial trifunctional protein (MTP) subunits were decreased in 2DE gels, immunoblotting identified their overall abundance as being differently dysregulated: in the gastric tumor samples, the MTP-α subunit was decreased, and the MTP-ß subunit was increased. On the other hand, heterogenous nuclear ribonucleoprotein M and galectin-4 were increased in the gastric tumor samples in both 2DE and immunoblotting.

The Global Sequence Signature algorithm unveils a structural network surrounding heavy chain CDR3 loop in Camelidae variable domains.

BACKGROUND: A large fraction of camelid (camels and llamas) antibodies is composed of heavy chain-only homodimers, able to recognise antigens with their variable domain. Events in somatic assembly and maturation of antibodies such as hypermutations and rearrangement of variable loops (CDRs - complementary determining regions) and selection among a wide range of framework variants are generally considered to be random processes. METHODS: An original algorithmic approach (Global Sequence Signature-GSS) was developed, able to take into account multiple functional and/or local sequence properties to detect scattered evolutionary constraints into sequences. RESULTS: Using the GSS approach, we show that the length of the main hypervariable loop (CDR3) is linked to the nature of 19 surrounding residues on the scaffold. Surprisingly, the relation between CDR3 size and scaffold residues strongly depends on the considered species, illustrating either significant differences in selection mechanisms or functional constraints during antibody maturation. CONCLUSIONS: Combined with the statistical coupling analysis (SCA) approach at the level of scaffold residues, this study has unravelled a robust interaction network on antibody structure surrounding the CDR3 loop. GENERAL SIGNIFICANCE: In addition to the general applicability of the GSS algorithm, which can bring together functional and sequence data to locate hot spots of constrained evolution, the relationship between CDR3 and scaffold discussed here should be taken into account in protein engineering when designing antibody libraries.

Proteomic approaches in biomarker discovery: new perspectives in cancer diagnostics.

Despite remarkable progress in proteomic methods, including improved detection limits and sensitivity, these methods have not yet been established in routine clinical practice. The main limitations, which prevent their integration into clinics, are high cost of equipment, the need for highly trained personnel, and last, but not least, the establishment of reliable and accurate protein biomarkers or panels of protein biomarkers for detection of neoplasms. Furthermore, the complexity and heterogeneity of most solid tumours present obstacles in the discovery of specific protein signatures, which could be used for early detection of cancers, for prediction of disease outcome, and for determining the response to specific therapies. However, cancer proteome, as the end-point of pathological processes that underlie cancer development and progression, could represent an important source for the discovery of new biomarkers and molecular targets for tailored therapies.

New assay for quantification of PEGylated proteins during in vitro permeability studies.

One of the major challenges when analyzing very low amounts of PEGylated proteins is finding a sensitive analytical method. Immunoassays are most frequently used, however, conjugation can partially or completely mask protein epitopes, which can substantially lower the response and influence the quantitation range. Here we describe a novel assay that allows quantification of low amounts of PEGylated or differently conjugated proteins. The basic principle is similar to the classic sandwich ELISA but there are no antibodies used neither for capture nor for detection. Instead, Ni(2+) chelation is exploited for capture and affinity between streptavidin and biotin for the detection step. The usefulness of the assay was proven in permeation studies (Caco-2 cell model) using diversely conjugated TNF-a protein. This approach could be extended to numerous other proteins eliminating the need to develop a separate assay for each protein/project.

Human granulocyte colony stimulating factor (G-CSF) produced in the filamentous fungus Aspergillus niger.

For the first time, a fungal production system is described for expression and secretion of the medically important human protein G-CSF, in Aspergillus niger. A reliable strategy was chosen with in-frame fusion of G-CSF behind a KEX2 cleavage site downstream of the coding region of the highly secreted homologous glucoamylase. This provided secretion levels of 5-10 mg/l culture medium of correctly processed G-CSF, although the majority of the protein (>90%) was biologically inactive. Following denaturation/ concentration and chromatographic separation/ renaturation, the G-CSF proliferation activity increased considerably, and analytical immobilised metal affinity chromatography confirmed the monomeric and correctly folded protein. These data suggest that this human secretory protein secreted into the medium of A. niger was not correctly folded, and that it escaped the endoplasmic reticulum folding control systems. This is compared to the folding of G-CSF produced in bacteria and yeast.

Recombinant human erythropoietin alters gene expression and stimulates proliferation of MCF-7 breast cancer cells.

BACKGROUND: Functional erythropoietin (EPO) signaling is not specific only to erythroid lineages and has been confirmed in several solid tumors, including breast. Three different isoforms of erythropoietin receptor (EPOR) have been reported, the soluble (EPOR-S) and truncated (EPOR-T) forms acting antagonistically to the functional EPOR. In this study, we investigated the effect of human recombinant erythropoietin (rHuEPO) on cell proliferation, early gene response and the expression of EPOR isoforms in the MCF-7 breast cancer cell line. MATERIALS AND METHODS: The MCF-7 cells were cultured with or without rHuEPO for 72 h or 10 weeks and assessed for their growth characteristics, expression of early response genes and different EPOR isoforms. The expression profile of EPOR and EPOR-T was determined in a range of breast cancer cell lines and compared with their invasive properties. RESULTS: MCF-7 cell proliferation after rHuEPO treatment was dependent on the time of treatment and the concentration used. High rHuEPO concentrations (40 U/ml) stimulated cell proliferation independently of a preceding long-term exposure of MCF-7 cells to rHuEPO, while lower concentrations increased MCF-7 proliferation only after 10 weeks of treatment. Gene expression analysis showed activation of EGR1 and FOS, confirming the functionality of EPOR. rHuEPO treatment also slightly increased the expression of the functional EPOR isoform, which, however, persisted throughout the 10 weeks of treatment. The expression levels of EPOR-T were not influenced. There were no correlations between EPOR expression and the invasiveness of MCF-7, MDA-MB-231, Hs578T, Hs578Bst, SKBR3, T-47D and MCF-10A cell lines. CONCLUSIONS: rHuEPO modulates MCF-7 cell proliferation in time- and concentration-dependent manner. We confirmed EGR1, FOS and EPOR as transcription targets of the EPO-EPOR signaling loop, but could not correlate the expression of different EPOR isoforms with the invasiveness of breast cancer cell lines.

Anti-Vimentin Nanobody Decreases Glioblastoma Cell Invasion In Vitro and In Vivo.

PURPOSE: Glioblastoma (GBM) is the most common primary brain tumour and one of the deadliest cancers. In addition to late diagnosis and inadequate treatment, the extremely low survival rate is also due to the lack of appropriate therapeutic biomarkers and corresponding therapeutic agents. One of the potential therapeutic biomarkers is the intermediate filament vimentin, which is associated with epithelial-mesenchymal transition (EMT). The purpose of this study was to analyse the effect of the anti-vimentin nanobody Nb79 on cell invasion in vitro and in vivo. To further our understanding of the mechanism of action, we investigated the association between Nb79 and EMT in GBM and GBM stem cells by analysing the expression levels of key EMT-related proteins. METHODS: The expression of vimentin in glioma tissues and cells was determined by RT-qPCR. An invasion assay was performed on differentiated glioblastoma cell line U-87 MG and stem cell line NCH421k in vitro as well as in vivo in zebrafish embryos. The effect of Nb79 on expression of EMT biomarkers beta-catenin, vimentin, ZEB-1 and ZO1 was determined by Western blot and immunocytochemistry. RESULTS: Our study shows that vimentin is upregulated in glioblastoma tissue compared to lower grade glioma and non-tumour brain tissue. We demonstrated that treatment with Nb79 reduced glioblastoma cell invasion by up to 64% in vitro and up to 21% in vivo. In addition, we found that the tight junction protein ZO-1 had higher expression on the cell membrane, when treated with inhibitory anti-vimentin Nb79 compared to control. CONCLUSION: In conclusion, our results suggest that anti-vimentin nanobody Nb79 is a promising tool to target glioblastoma cell invasion.

The versatility of the fungal cytochrome P450 monooxygenase system is instrumental in xenobiotic detoxification.

Cytochromes P450 (CYPs) catalyse diverse reactions and are key enzymes in fungal primary and secondary metabolism, and xenobiotic detoxification. CYP enzymatic properties and substrate specificity determine the reaction outcome. However, CYP-mediated reactions may also be influenced by their redox partners. Filamentous fungi with numerous CYPs often possess multiple microsomal redox partners, cytochrome P450 reductases (CPRs). In the plant pathogenic ascomycete Cochliobolus lunatus we recently identified two CPR paralogues, CPR1 and CPR2. Our objective was to functionally characterize two endogenous fungal cytochrome P450 systems and elucidate the putative physiological roles of CPR1 and CPR2. We reconstituted both CPRs with CYP53A15, or benzoate 4-hydroxylase from C. lunatus, which is crucial in the detoxification of phenolic plant defence compounds. Biochemical characterization using RP-HPLC shows that both redox partners support CYP activity, but with different product specificities. When reconstituted with CPR1, CYP53A15 converts benzoic acid to 4-hydroxybenzoic acid, and 3-methoxybenzoic acid to 3-hydroxybenzoic acid. However, when the redox partner is CPR2, both substrates are converted to 3,4-dihydroxybenzoic acid. Deletion mutants and gene expression in mycelia grown on media with inhibitors indicate that CPR1 is important in primary metabolism, whereas CPR2 plays a role in xenobiotic detoxification.

Inclusion bodies as potential vehicles for recombinant protein delivery into epithelial cells.

BACKGROUND: We present the potential of inclusion bodies (IBs) as a protein delivery method for polymeric filamentous proteins. We used as cell factory a strain of E. coli, a conventional host organism, and keratin 14 (K14) as an example of a complex protein. Keratins build the intermediate filament cytoskeleton of all epithelial cells. In order to build filaments, monomeric K14 needs first to dimerize with its binding partner (keratin 5, K5), which is then followed by heterodimer assembly into filaments. RESULTS: K14 IBs were electroporated into SW13 cells grown in culture together with a "reporter" plasmid containing EYFP labeled keratin 5 (K5) cDNA. As SW13 cells do not normally express keratins, and keratin filaments are built exclusively of keratin heterodimers (i.e. K5/K14), the short filamentous structures we obtained in this study can only be the result of: a) if both IBs and plasmid DNA are transfected simultaneously into the cell(s); b) once inside the cells, K14 protein is being released from IBs; c) released K14 is functional, able to form heterodimers with EYFP-K5. CONCLUSIONS: Soluble IBs may be also developed for complex cytoskeletal proteins and used as nanoparticles for their delivery into epithelial cells.

Virtual screening yields inhibitors of novel antifungal drug target, benzoate 4-monooxygenase.

Fungal CYP53 enzymes are highly conserved proteins, involved in phenolic detoxification, and have no homologues in higher eukaryotes, rendering them favorable drug targets. Aiming to discover novel CYP53 inhibitors, we employed two parallel virtual screening protocols and evaluated highest scoring hit compounds by analyzing the spectral binding interactions, by surveying the antifungal activity, and assessing the inhibition of catalytic activity. On the basis of combined results, we selected 3-methyl-4-(1H-pyrrol-1-yl)benzoic acid (compound 2) as the best candidate for hit-to-lead follow-up in the antifungal drug discovery process.