A Monocarbonyl Curcuminoid Derivative Inhibits the Activity of Human Glutathione Transferase A4-4 and Chemosensitizes Glioblastoma Cells to Temozolomide.

Human glutathione transferase A4-4 (hGSTA4-4) displays high catalytic efficiency towards 4-hydroxyalkenals and other cytotoxic and mutagenic products of radical reactions and lipid peroxidation. Its role as a target for the chemosensitization of cancer cells has not been investigated so far. In this study, the inhibitory potency of twelve selected natural products and ten monocarbonyl curcumin derivatives against hGSTA4-4 was studied. Among natural products, ellagic acid turned out to be the strongest inhibitor with an IC50 value of 0.44 ± 0.01 µM. Kinetic analysis using glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB) as variable substrates showed that ellagic acid behaved as a competitive inhibitor towards both GSH and CDNB, with Ki values of 0.39 ± 0.02 and 0.63 ± 0.03 µM, respectively. Among the curcumin derivatives studied, three proved to be the most potent inhibitors, in the order DM151 > DM101 > DM100, with IC50 values of 2.4 ± 0.1 µM, 12.7 ± 1.1 µΜ and 16.9 ± 0.4 µΜ, respectively. Further kinetic inhibition analysis of the most active derivative, DM151, demonstrated that this compound is a mixed inhibitor towards CDNB with inhibition constants of Ki = 4.1 ± 0.5 µM and Ki' = 0.536 ± 0.034 µM, while it is a competitive inhibitor towards GSH with a Ki = 0.98 ± 0.11 µM. Molecular docking studies were performed to interpret the differences in binding of ellagic acid and curcumin derivatives to hGSTA4-4. The in silico measured docking scores were consistent with the obtained experimental data. Hydrogen bonds appear to be the main contributors to the specific binding of monocarbonyl curcumin derivatives, while π-π stacking interactions play a key role in the enzyme-ellagic acid interaction. In vitro cytotoxicity assessment of the worst (DM148) and the best (DM151) inhibitors was performed against glioblastoma cell lines U-251 MG and U-87 MG. The results revealed that DM151 displays considerably higher cytotoxicity against both glioblastoma cell lines, while the glioblastoma cytotoxicity of DM148 was very limited. Furthermore, low and non-toxic doses of DM151 sensitized U-251 MG cells to the first-line glioblastoma chemotherapeutic temozolomide (TMZ), allowing us to propose for the first time that hGSTA4-4 inhibitors may be attractive therapeutic partners for TMZ to optimize its clinical effect in glioblastoma chemotherapy.

Metagenomic analysis of hot spring soil for mining a novel thermostable enzybiotic.

The misuse and overuse of antibiotics have contributed to a rapid emergence of antibiotic-resistant bacterial pathogens. This global health threat underlines the urgent need for innovative and novel antimicrobials. Endolysins derived from bacteriophages or prophages constitute promising new antimicrobials (so-called enzybiotics), exhibiting the ability to break down bacterial peptidoglycan (PG). In the present work, metagenomic analysis of soil samples, collected from thermal springs, allowed the identification of a prophage-derived endolysin that belongs to the N-acetylmuramoyl-L-alanine amidase type 2 (NALAA-2) family and possesses a LysM (lysin motif) region as a cell wall binding domain (CWBD). The enzyme (Ami1) was cloned and expressed in Escherichia coli, and its bactericidal and lytic activity was characterized. The results indicate that Ami1 exhibits strong bactericidal and antimicrobial activity against a broad range of bacterial pathogens, as well as against isolated peptidoglycan (PG). Among the examined bacterial pathogens, Ami1 showed highest bactericidal activity against Staphylococcus aureus sand Staphylococcus epidermidis cells. Thermostability analysis revealed a melting temperature of 64.2 ± 0.6 °C. Overall, these findings support the potential that Ami1, as a broad spectrum antimicrobial agent, could be further assessed as enzybiotic for the effective treatment of bacterial infections. KEY POINTS: • Metagenomic analysis allowed the identification of a novel prophage endolysin • The endolysin belongs to type 2 amidase family with lysin motif region • The endolysin displays high thermostability and broad bactericidal spectrum.

Microalgae as a Sustainable Source of Antioxidants in Animal Nutrition, Health and Livestock Development.

Microalgae are a renewable and sustainable source of bioactive compounds, such as essential amino acids, polyunsaturated fatty acids, and antioxidant compounds, that have been documented to have beneficial effects on nutrition and health. Among these natural products, the demand for natural antioxidants, as an alternative to synthetic antioxidants, has increased. The antioxidant activity of microalgae significantly varies between species and depends on growth conditions. In the last decade, microalgae have been explored in livestock animals as feed additives with the aim of improving both animals' health and performance as well as product quality and the environmental impact of livestock. These findings are highly dependent on the composition of microalgae strain and their amount in the diet. The use of carbohydrate-active enzymes can increase nutrient bioavailability as a consequence of recalcitrant microalgae cell wall degradation, making it a promising strategy for monogastric nutrition for improving livestock productivity. The use of microalgae as an alternative to conventional feedstuffs is becoming increasingly important due to food-feed competition, land degradation, water deprivation, and climate change. However, the cost-effective production and use of microalgae is a major challenge in the near future, and their cultivation technology should be improved by reducing production costs, thus increasing profitability.

Metaviromics analysis of marine biofilm reveals a glycoside hydrolase endolysin with high specificity towards Acinetobacter baumannii.

Multidrug-resistant (MDR) bacteria are a growing threat to the public health. Among them, the Gram-negative Acinetobacter baumannii is considered today as the most dangerous MDR pathogen. Phage-derived endolysins are peptidoglycan (PG) hydrolytic enzymes that can function as effective tools in the fight against MDR bacteria. In the present work, the viral diversity of a marine environmental sample (biofilm), formed near an industrial zone, was mined for the identification of a putative endolysin (AbLys2) that belongs to the glycoside hydrolase family 24 (GH24, EC 3.2.1.17). The coding sequence of AbLys2 was cloned and expressed in E. coli. The lytic activity and specificity of the recombinant enzyme were evaluated against suspensions of a range of Gram-positive and Gram-negative human pathogens using turbidity assays. AbLys2 displayed enhanced selectivity towards A. baumannii cells, compared to other bacteria. Kinetics analysis was carried out to characterize the dependence of its lytic activity on pH and showed that the enzyme exhibits its maximal activity at pH 5.5. Thermostability analysis showed that AbLys2 displays melting temperature Tm 47.1 °C. Florescence microscopy and cell viability assays established that AbLys2 is active towards live cultures of A. baumannii cells with an inhibitory concentration IC50 3.41 ± 0.09 µM. Molecular modeling allowed the prediction of important amino acid residues involved in catalysis. The results of the present study suggest that AbLys2 provides efficient lytic and antimicrobial activity towards A. baumannii cells and therefore is a promising new antimicrobial against this pathogen.

Structural and functional features of a broad-spectrum prophage-encoded enzybiotic from Enterococcus faecium.

Multidrug-resistant (MDR) bacteria have become a growing threat to public health. The gram-positive Enterococcus faecium is classified by WHO as a high-priority pathogen among the global priority list of antibiotic-resistant bacteria. Peptidoglycan-degrading enzymes (PDEs), also known as enzybiotics, are useful bactericidal agents in the fight against resistant bacteria. In this work, a genome-based screening approach of the genome of E. faecium allowed the identification of a putative PDE gene with predictive amidase activity (EfAmi1; EC 3.5.1.28) in a prophage-integrated sequence. EfAmi1 is composed by two domains: a N-terminal Zn2+-dependent N-acetylmuramoyl-L-alanine amidase-2 (NALAA-2) domain and a C-terminal domain with unknown structure and function. The full-length gene of EfAmi1 was cloned and expressed as a 6xHis-tagged protein in E. coli. EfAmi1 was produced as a soluble protein, purified, and its lytic and antimicrobial activities were investigated using turbidity reduction and Kirby-Bauer disk-diffusion assays against clinically isolated bacterial pathogens. The crystal structure of the N-terminal amidase-2 domain was determined using X-ray crystallography at 1.97 Å resolution. It adopts a globular fold with several α-helices surrounding a central five-stranded ß-sheet. Sequence comparison revealed a cluster of conserved amino acids that defines a putative binding site for a buried zinc ion. The results of the present study suggest that EfAmi1 displays high lytic and antimicrobial activity and may represent a promising new antimicrobial in the post-antibiotic era.

Inhibition Analysis and High-Resolution Crystal Structure of Mus musculus Glutathione Transferase P1-1.

Multidrug resistance is a significant barrier that makes anticancer therapies less effective. Glutathione transferases (GSTs) are involved in multidrug resistance mechanisms and play a significant part in the metabolism of alkylating anticancer drugs. The purpose of this study was to screen and select a lead compound with high inhibitory potency against the isoenzyme GSTP1-1 from Mus musculus (MmGSTP1-1). The lead compound was selected following the screening of a library of currently approved and registered pesticides that belong to different chemical classes. The results showed that the fungicide iprodione [3-(3,5-dichlorophenyl)-2,4-dioxo-N-propan-2-ylimidazolidine-1-carboxamide] exhibited the highest inhibition potency (ΙC50 = 11.3 ± 0.5 µΜ) towards MmGSTP1-1. Kinetics analysis revealed that iprodione functions as a mixed-type inhibitor towards glutathione (GSH) and non-competitive inhibitor towards 1-chloro-2,4-dinitrobenzene (CDNB). X-ray crystallography was used to determine the crystal structure of MmGSTP1-1 at 1.28 Å resolution as a complex with S-(p-nitrobenzyl)glutathione (Nb-GSH). The crystal structure was used to map the ligand-binding site of MmGSTP1-1 and to provide structural data of the interaction of the enzyme with iprodione using molecular docking. The results of this study shed light on the inhibition mechanism of MmGSTP1-1 and provide a new compound as a potential lead structure for future drug/inhibitor development.

Characterization and Engineering Studies of a New Endolysin from the Propionibacterium acnes Bacteriophage PAC1 for the Development of a Broad-Spectrum Artilysin with Altered Specificity.

The emergence of multidrug-resistant (MDR) bacteria has risen rapidly, leading to a great threat to global public health. A promising solution to this problem is the exploitation of phage endolysins. In the present study, a putative N-acetylmuramoyl-L-alanine type-2 amidase (NALAA-2, EC 3.5.1.28) from Propionibacterium bacteriophage PAC1 was characterized. The enzyme (PaAmi1) was cloned into a T7 expression vector and expressed in E. coli BL21 cells. Kinetics analysis using turbidity reduction assays allowed the determination of the optimal conditions for lytic activity against a range of Gram-positive and negative human pathogens. The peptidoglycan degradation activity of PaAmi1 was confirmed using isolated peptidoglycan from P. acnes. The antibacterial activity of PaAmi1 was investigated using live P. acnes cells growing on agar plates. Two engineered variants of PaAmi1 were designed by fusion to its N-terminus two short antimicrobial peptides (AMPs). One AMP was selected by searching the genomes of Propionibacterium bacteriophages using bioinformatics tools, whereas the other AMP sequence was selected from the antimicrobial peptide databases. Both engineered variants exhibited improved lytic activity towards P. acnes and the enterococci species Enterococcus faecalis and Enterococcus faecium. The results of the present study suggest that PaAmi1 is a new antimicrobial agent and provide proof of concept that bacteriophage genomes are a rich source of AMP sequences that can be further exploited for designing novel or improved endolysins.

A Key Role in Catalysis and Enzyme Thermostability of a Conserved Helix H5 Motif of Human Glutathione Transferase A1-1.

Glutathione transferases (GSTs) are promiscuous enzymes whose main function is the detoxification of electrophilic compounds. These enzymes are characterized by structural modularity that underpins their exploitation as dynamic scaffolds for engineering enzyme variants, with customized catalytic and structural properties. In the present work, multiple sequence alignment of the alpha class GSTs allowed the identification of three conserved residues (E137, K141, and S142) at α-helix 5 (H5). A motif-directed redesign of the human glutathione transferase A1-1 (hGSTA1-1) was performed through site-directed mutagenesis at these sites, creating two single- and two double-point mutants (E137H, K141H, K141H/S142H, and E137H/K141H). The results showed that all the enzyme variants displayed enhanced catalytic activity compared to the wild-type enzyme hGSTA1-1, while the double mutant hGSTA1-K141H/S142H also showed improved thermal stability. X-ray crystallographic analysis revealed the molecular basis of the effects of double mutations on enzyme stability and catalysis. The biochemical and structural analysis presented here will contribute to a deeper understanding of the structure and function of alpha class GSTs.

Characterization of a glycoside hydrolase endolysin from Acinetobacter baumannii phage AbTZA1 with high antibacterial potency and novel structural features.

The classification of Acinetobacter baumannii by WHO as 'priority 1' antibiotic-resistant pathogen underlines the urgent need for novel antimicrobial agents towards this pathogen. In this work, screening of the A. baumannii phage AbTZA1 genome allowed the identification of a putative endolysin (AbLys1, EC3.2.1.17) that belongs to the glycoside hydrolase family 24 (GH24). The sequence of AbLys1 was cloned, expressed in E. coli and purified. The lytic activity and specificity of AbLys1 were evaluated against a range of Gram-positive and Gram-negative human pathogens. AbLys1 was found to display a high selectivity towards A. baumannii. Kinetic analysis was carried out to characterize the dependence of its lytic activity on pH. The enzyme shows its maximal activity at pH values 7-8. The structure of AbLys1 was determined by X-ray crystallography to 1.82 Å resolution. The overall structure revealed two helical domains: a small, antenna-like, N-terminal domain and a larger C-terminal domain with six α-helices and a ß-hairpin. Both the antenna-like and ß-hairpin regions contain short sequences (AMseq1 and AMseq2) with predicted antimicrobial activity. Engineering studies revealed a key role of AMseq1 and AMseq2 on the enzyme's lytic activity towards A. baumannii cells but not towards purified peptidoglycan. This suggests that both sequences affect the destabilization of the outer membrane, thus providing access of the catalytic domain to the peptidoglycan. In addition, the deletion of AMseq1 enhanced the enzyme stability, whereas the deletion of AMseq2 diminished it. The results suggest that AbLys1 is a promising new enzybiotic with efficient lytic and antimicrobial activity.

Structural Studies of Klebsiella pneumoniae Fosfomycin-Resistance Protein and Its Application for the Development of an Optical Biosensor for Fosfomycin Determination.

Fosfomycin-resistance proteins (FosAs) are dimeric metal-dependent glutathione transferases that conjugate the antibiotic fosfomycin (Fos) to the tripeptide glutathione (γ-Glu-Cys-Gly, GSH), rendering it inactive. In the present study, we reported a comparative analysis of the functional features of two FosAs from Pseudomonas aeruginosa (FosAPA) and Klebsiella pneumoniae (FosAKP). The coding sequences of the enzymes were cloned into a T7 expression vector, and soluble active enzymes were expressed in E. coli. FosAKP displayed higher activity and was selected for further studies. The crystal structure of the dimeric FosAKP was determined via X-ray crystallography at 1.48 Šresolution. Fos and tartrate (Tar) were found bound in the active site of the first and second molecules of the dimer, respectively. The binding of Tar to the active site caused slight rearrangements in the structure and dynamics of the enzyme, acting as a weak inhibitor of Fos binding. Differential scanning fluorimetry (DSF) was used to measure the thermal stability of FosAKP under different conditions, allowing for the selection of a suitable buffer to maximize enzyme operational stability. FosAKP displays absolute specificity towards Fos; therefore, this enzyme was exploited for the development of an enzyme-based colorimetric biosensor. FosAKP was tethered at the bottom of a plastic cuvette using glutaraldehyde chemistry to develop a simple colorimetric method for the determination of Fos in drinking water and animal plasma.

Repurposing Glutathione Transferases: Directed Evolution Combined with Chemical Modification for the Creation of a Semisynthetic Enzyme with High Hydroperoxidase Activity.

Glutathione peroxidases (GPXs) are antioxidant selenoenzymes, which catalyze the reduction of hydroperoxides via glutathione (GSH), providing protection to cells against oxidative stress metabolites. The present study aims to create an efficient semisynthetic GPX based on the scaffold of tau class glutathione transferase (GSTU). A library of GSTs was constructed via DNA shuffling, using three homologue GSTUs from Glycine max as parent sequences. The DNA library of the shuffled genes was expressed in E. coli and the catalytic activity of the shuffled enzymes was screened using cumene hydroperoxide (CuOOH) as substrate. A chimeric enzyme variant (named Sh14) with 4-fold enhanced GPX activity, compared to the wild-type enzyme, was identified and selected for further study. Selenocysteine (Sec) was substituted for the active-site Ser13 residue of the Sh14 variant via chemical modification. The GPX activity (kcat) and the specificity constant (kcat/Κm) of the evolved seleno-Sh14 enzyme (SeSh14) was increased 177- and 2746-fold, respectively, compared to that of the wild-type enzyme for CuOOH. Furthermore, SeSh14 effectively catalyzed the reduction of hydrogen peroxide, an activity that is completely undetectable in all GSTs. Such an engineered GPX-like biocatalyst based on the GSTU scaffold might serve as a catalytic bioscavenger for the detoxification of hazardous hydroperoxides. Furthermore, our results shed light on the evolution of GPXs and their structural and functional link with GSTs.

Coumarin-Encapsulated Solid Lipid Nanoparticles as an Effective Therapy against Methicillin-Resistant Staphylococcus aureus.

Bacterial infections caused by antibiotic-resistant pathogens are a significant public health problem. This is because the transmission of infectious diseases is shifting, and new antibiotic-resistant strains of bacteria are emerging. The development of biofilms that are resistant to antibiotics poses another hurdle to drugs and treatment alternatives. Therefore, there is an urgent need to develop innovative strategies to effectively eliminate antibiotic-resistant microorganisms effectively. Natural coumarins have broad spectrum bioactivity and the potential for lower resistance. Coumarin is a secondary metabolite found in certain plants, fungi, and bacteria. It is highly effective against methicillin-resistant Staphylococcus aureus (MRSA). Therefore, coumarin can be used as an alternative to combat MRSA. However, most antibacterial agents lack selective targeting of pathological sites, limiting the efficacy of their antibacterial activity. Efficient MRSA treatments can be achieved through nanoparticle (NPs)-based targeted therapies. To address this challenge, a novel coumarin-loaded solid lipid nanocarrier for MRSA was developed to overcome this challenge. The developed systems exhibited a particle size of 138.5 ± 76.06 nm and a polydispersity index (PDI) of 0.245 ± 0.00. The zeta potential of coumarin-loaded SLNs was reported to be -22.2 ± 8.15 mV with a spherical shape. The encapsulation efficiency of coumarin was reported to be 63.09 ± 3.46% in the final formulation. The developed formulation was biocompatible with a minimum inhibitory concentration (MIC) of 1.08 µg/mL. This study suggests that coumarin-loaded SLNs can effectively treat MRSA infections.

Myricetin as a Potential Adjuvant in Chemotherapy: Studies on the Inhibition of Human Glutathione Transferase A1-1.

Glutathione transferases (GSTs) are a family of Phase II detoxification enzymes that are involved in the development of multi-drug resistance (MDR) phenomena toward chemotherapeutic agents. GST inhibitors are considered candidate compounds able to chemomodulate and reverse MDR. The natural flavonoid myricetin (MYR) has been shown to exhibit a wide range of pharmacological functions, including antitumor activity. In the present work, the interaction of MYR with human glutathione transferase A1-1 (hGSTA1-1) was investigated by kinetics inhibition analysis and molecular modeling studies. The results showed that MYR binds with high affinity to hGSTA1-1 (IC50 2.1 ± 0.2 µΜ). It functions as a non-competitive inhibitor towards the electrophile substrate 1-chloro-2,4-dinitrobenzene (CDNB) and as a competitive inhibitor towards glutathione (GSH). Chemical modification studies with the irreversible inhibitor phenethyl isothiocyanate (PEITC), in combination with in silico molecular docking studies allowed the prediction of the MYR binding site. MYR appears to bind at a distinct location, partially overlapping the GSH binding site (G-site). The results of the present study show that MYR is a potent inhibitor of hGSTA1-1 that can be further exploited towards the development of natural, safe, and effective GST-targeted cancer chemosensitizers.

Development of Gum-Acacia-Stabilized Silver Nanoparticles Gel of Rutin against Candida albicans.

Candida spp. is one of the most causative pathogens responsible for fungal infections. It is often a hospital-acquired form of sepsis with a very high number of deaths. Currently, the most effective anti-fungal agents are based on polyenes or echinocandins. However, long-term treatments or repeated use of these anti-fungals lead to therapy limitations. Current research is urgently needed to overcome existing challenges for antimicrobials from natural sources. This study aims to determine the anti-fungal activity of rutin, which has the advantage of increasing the therapeutic value. Because of its low solubility in water and oils, rutin is limited in use. To address these constraints, we encapsulated rutin in a nanocarrier system. Silver nanoparticles (SNPs) and gum acacia (GAs) are emerging as attractive components and are widely studied as biologically safe nanomaterials/carrier systems for various drugs. Still, they are barely investigated as nano-sized vectors for the targeted delivery of rutin. In the present work, GA stabilised SNPs of rutin were successfully formulated and evaluated. It was later incorporated into carbapol 940 gels and formed SNP gels. Rutin-SNPs were developed with a consistent size in the nano range of 59.67 ± 44.24 nm in size, 0.295 ± 0.014 polydispersity index (PDI), and -11.2 ± 6.66 mV zeta potential. The drug released was found to be 81. 26 ± 4.06% in 600 min by following zero-order kinetics. The rutin-SNP gel showed considerable activity against C. albicans skin candidiasis at MIC 1.56 g/mL. The developed formulation was biocompatible. This first-ever interdisciplinary study suggests that the rutin-SNPs gel could play a vital role in drug resistance in this fungal pathogen.

Directed Evolution of Phi Class Glutathione Transferases Involved in Multiple-Herbicide Resistance of Grass Weeds and Crops.

The extensive application of herbicides in crop cultivation has indisputably led to the emergence of weed populations characterized by multiple herbicide resistance (MHR). This phenomenon is associated with the enhanced metabolism and detoxifying ability of endogenous enzymes, such as phi class glutathione transferases (GSTFs). In the present work, a library of mutant GSTFs was created by in vitro directed evolution via DNA shuffling. Selected gstf genes from the weeds Alopecurus myosuroides and Lolium rigidum, and the cereal crops Triticum durum and Hordeum vulgare were recombined to forge a library of novel chimeric GSTFs. The library was activity screened and the best-performing enzyme variants were purified and characterized. The work allowed the identification of enzyme variants that exhibit an eight-fold improvement in their catalytic efficiency, higher thermal stability (8.3 °C) and three-times higher inhibition sensitivity towards the herbicide butachlor. The crystal structures of the best-performing enzyme variants were determined by X-ray crystallography. Structural analysis allowed the identification of specific structural elements that are responsible for kcat regulation, thermal stability and inhibition potency. These improved novel enzymes hold the potential for utilization in biocatalysis and green biotechnology applications. The results of the present work contribute significantly to our knowledge of the structure and function of phi class plant GSTs and shed light on their involvement in the mechanisms of MHR.

Rational Design of Self-Assembling Supramolecular Protein Nanostructures Utilizing the Cucurbit[8]Uril Macrocyclic Host.

Self-assembly is a phenomenon that governs molecular structural organization in nature, therefore raising research interest for the fabrication of novel nanomaterials with diverse applications in biocatalysis, biomedicine, material templating, and biosensor development. In this chapter we provide protocols for the development of supramolecular protein complexes based on host-guest interactions in the presence of the macrocyclic host, cucurbit[8]uril (CB[8]). CB[8] is reported to exhibit high binding affinity towards the tripeptide Phe-Gly-Gly (FGG), therefore it can be utilized as a selective adhesive of protein molecules, after fusion of FGG to an accessible protein surface.

Ligandability Assessment of Human Glutathione Transferase M1-1 Using Pesticides as Chemical Probes.

Glutathione transferases (GSTs; EC 2.5.1.18) form a group of multifunctional enzymes that are involved in phase II of the cellular detoxification mechanism and are associated with increased susceptibility to cancer development and resistance to anticancer drugs. The present study aims to evaluate the ligandability of the human GSTM1-1 isoenzyme (hGSTM1-1) using a broad range of structurally diverse pesticides as probes. The results revealed that hGSTM1-1, compared to other classes of GSTs, displays limited ligandability and ligand-binding promiscuity, as revealed by kinetic inhibition studies. Among all tested pesticides, the carbamate insecticide pirimicarb was identified as the strongest inhibitor towards hGSTM1-1. Kinetic inhibition analysis showed that pirimicarb behaved as a mixed-type inhibitor toward glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB). To shine a light on the restricted hGSTM1-1 ligand-binding promiscuity, the ligand-free crystal structure of hGSTM1-1 was determined by X-ray crystallography at 1.59 Å-resolution. Comparative analysis of ligand-free structure with the available ligand-bound structures allowed for the study of the enzyme's plasticity and the induced-fit mechanism operated by hGSTM1-1. The results revealed important structural features of the H-site that contribute to xenobiotic-ligand binding and specificity. It was concluded that hGSTM1-1 interacts preferentially with one-ring aromatic compounds that bind at a discrete site which partially overlaps with the xenobiotic substrate binding site (H-site). The results of the study form a basis for the rational design of new drugs targeting hGSTM1-1.

Structural and Functional Characterization of Camelus dromedarius Glutathione Transferase M1-1.

Glutathione transferases (GSTs; EC. 2.5.1.18) are a large family of multifunctional enzymes that play crucial roles in the metabolism and inactivation of a broad range of xenobiotic compounds. In the present work, we report the kinetic and structural characterization of the isoenzyme GSTM1-1 from Camelus dromedarius (CdGSTM1-1). The CdGSΤM1-1 was expressed in E. coli BL21 (DE3) and was purified by affinity chromatography. Kinetics analysis showed that the enzyme displays a relative narrow substrate specificity and restricted ability to bind xenobiotic compounds. The crystal structures of CdGSΤM1-1 were determined by X-ray crystallography in complex with the substrate (GSH) or the reaction product (S-p-nitrobenzyl-GSH), providing snapshots of the induced-fit catalytic mechanism. The thermodynamic stability of CdGSTM1-1 was investigated using differential scanning fluorimetry (DSF) in the absence and in presence of GSH and S-p-nitrobenzyl-GSH and revealed that the enzyme's structure is significantly stabilized by its ligands. The results of the present study advance the understanding of camelid GST detoxification mechanisms and their contribution to abiotic stress adaptation in harsh desert conditions.

Monocarbonyl Curcumin Analogues as Potent Inhibitors against Human Glutathione Transferase P1-1.

The isoenzyme of human glutathione transferase P1-1 (hGSTP1-1) is involved in multi-drug resistance (MDR) mechanisms in numerous cancer cell lines. In the present study, the inhibition potency of two curcuminoids and eleven monocarbonyl curcumin analogues against hGSTP1-1 was investigated. Demethoxycurcumin (Curcumin II) and three of the monocarbonyl curcumin analogues exhibited the highest inhibitory activity towards hGSTP1-1 with IC50 values ranging between 5.45 ± 1.08 and 37.72 ± 1.02 µM. Kinetic inhibition studies of the most potent inhibitors demonstrated that they function as non-competitive/mixed-type inhibitors. These compounds were also evaluated for their toxicity against the prostate cancer cells DU-145. Interestingly, the strongest hGSTP1-1 inhibitor, (DM96), exhibited the highest cytotoxicity with an IC50 of 8.60 ± 1.07 µΜ, while the IC50 values of the rest of the compounds ranged between 44.59-48.52 µΜ. Structural analysis employing molecular docking, molecular dynamics (MD) simulations, and binding-free-energy calculations was performed to study the four most potent curcumin analogues as hGSTP1-1 inhibitors. According to the obtained computational results, DM96 exhibited the lowest binding free energy, which is in agreement with the experimental data. All studied curcumin analogues were found to form hydrophobic interactions with the residue Gln52, as well as hydrogen bonds with the nearby residues Gln65 and Asn67. Additional hydrophobic interactions with the residues Phe9 and Val36 as well as π-π stacking interaction with Phe9 contributed to the superior inhibitory activity of DM96. The van der Waals component through shape complementarity was found to play the most important role in DM96-inhibitory activity. Overall, our results revealed that the monocarbonyl curcumin derivative DM96 acts as a strong hGSTP1-1 inhibitor, exerts high prostate cancer cell cytotoxicity, and may, therefore, be exploited for the suppression and chemosensitization of cancer cells. This study provides new insights into the development of safe and effective GST-targeted cancer chemosensitizers.