Correction: Isolation and molecular characterization of novel glucarpidases: Enzymes to improve the antibody directed enzyme pro-drug therapy for cancer treatment.

[This corrects the article DOI: 10.1371/journal.pone.0196254.].

Investigation on CT-DNA and Protein Interaction of New Pd(II) Complexes Involving Ceftazidime and 3-Amino-1,2,3-triazole: Synthesis, Characterization, Biological Impact, Anticancer Evaluation, and Molecular Docking Approaches.

Two new palladium (II) complexes, [Pd(CAZ)(OH2 )2 ]2+ (1) and [Pd(3-AT)(OH2 )2 ]2+ (2), (CAZ=ceftazidime, and 3-AT=amitrole) were synthesized and studied for their potential as anticancer drugs with low toxicity and high potency. To fully characterize these complexes, we conducted elemental analysis and FT-IR studies. Furthermore, we irradiated the complexes with Indian 60 Co gamma rays and thoroughly evaluated their antimicrobial properties. Our results demonstrate that the inhibitory activity of complexes was significantly enhanced against (G+) bacteria and fungi. Additionally, we probed the complexes' interaction with CT-DNA and BSA using various techniques, including UV-vis spectroscopy, thermal denaturation, viscometry, gel electrophoresis, and molecular docking studies. Our findings conclusively demonstrate that these complexes possess a strong binding interaction with CT-DNA via minor groove binding and/or electrostatic interactions, as well as excellent binding affinity to BSA. Finally, we conducted a cytotoxicity assay that clearly indicates these complexes hold immense promise as cell growth inhibitors against MCF-7 and HCT-116.

Antimicrobial screening involving Helicobacter pylori of nano-therapeutic compounds based on the amoxicillin antibiotic drug.

Nano-structure Cu(II) complex [Cu(AMAB)2 ]Cl2 with Schiff base (AMAB) derived from the condensation between 4-(dimethylamino)benzaldehyde and amoxicillin trihydrate was prepared. (AMAB) Schiff base and its Cu(II) complex were identified and confirmed by different physicochemical techniques. The Schiff base (AMAB) was coordinated to copper ion through carbonyl oxygen and imine nitrogen donor sites. X-ray powder diffraction shows a cubic crystal system of the Cu(II) complex. The density functional theory was used to optimize the structure geometries of the investigated compounds. The molecular docking of the active amino acids of the investigated proteins' interactions with the tested compounds was evaluated. The bactericidal or bacteriostatic effect of the compounds was screened against some bacterial strains. The activity of Cu-chelate against Gram-negative bacteria was mainly more effective than its (AMAB) ligand and vice versa in the case of Gram-positive bacteria. The biological activity of the prepared compounds with biomolecules calf thymus DNA (CT-DNA) was determined by electronic absorption spectra and DNA gel electrophoresis technique. All studies revealed that the Cu-chelate derivative exhibited better binding affinity to both CT-DNA than the AMAB and amoxicillin itself. The anti-inflammatory effect of the designed compounds was determined by testing their protein denaturation inhibitory activity spectrophotometrically. All obtained data supported that the designed nano-Cu(II) complex with Schiff base (AMAB) is a potent bactericide against H. pylori, and exhibits anti-inflammatory activity. The dual inhibition effects of the designed compound represent a modern therapeutic approach with extended spectrum of action. Therefore, it can act as good drug target in antimicrobial and anti-inflammtory therapies. Finally, H. pylori resistance to amoxicillin is absent or rare in many countries, thus amoxicillin nanoparticles may be beneficial for countries where amoxicillin resistance is reported.

Synthesis, Characterization, and Biological Evaluation of Cu(II) Complexes Containing Triflupromazine with Glycine and Histidine.

Two novel copper (II) complexes [Cu(TFP)(Gly)Cl] ⋅ 2H2 O complex (1) and [Cu(TFP)(His)Cl] ⋅ 2H2 O complex (2) are synthesized, where TFP stands for trifluropromazine, Gly. represents glycine, and His. is histidine. Chemical composition, IR, mass spectra, and magnetic susceptibility tests are performed. Complex binding with macromolecules was investigated using UV-vis, viscosity, gel electrophoresis, and fluorescence quenching. Fluorescence spectroscopy revealed that each complex could replace ethidium bromide (EB). These complexes exhibit grooved, non-covalent, and electrostatic interactions with CT-DNA. Spectroscopy analysis of the BSA interaction showed that complexes bind to protein (Kb values for (1) is 5.89×103  M-1 and for (2) is 9.08×103  M-1 ) more strongly than CT-DNA (Kb values for (1) is 5.43×103  M-1 and for (2) is 7.17×103  M-1 ). Molecular docking analysis and spectral absorption measurements showed high agreement. Antimicrobial, antioxidant, and anti-inflammatory properties were tested in vitro. The druggability of complex (2) should be tested in vivo as it is more biologically active.

An in vitro cytotoxicity of glufosfamide in HepG2 cells relative to its nonconjugated counterpart.

BACKGROUND: Glufosfamide (ß-D-glucosylisophosphoramide mustard, GLU) is an alkylating cytotoxic agent in which ifosforamide mustard (IPM) is glycosidically linked to the ß-D-glucose molecule. GLU exerted its cytotoxic effect as a targeted chemotherapy. Although, its cytotoxic efficacy in a number of cell lines, there were no experimental or clinical data available on the oncolytic effect of oxazaphosphorine drugs in hepatocellular carcinoma. Therefore, the main objective of the current study is to assess the cytotoxic potential of GLU for the first time in the hepatocellular carcinoma HepG2 cell line model. METHODS: Cytotoxicity was assayed by the MTT method, and half-maximal inhibitory concentration (IC50) was calculated. Flow cytometric analysis of apoptosis frequencies was measured by using Annexin V/PI double stain, an immunocytochemical assay of caspase-9, visualization of caspase-3, and Bcl2 gene expression were undertaken as apoptotic markers. Mitochondrial membrane potential was measured using the potentiometric dye; JC-1, as a clue for early apoptosis as well as ATP production, was measured by the luciferase-chemiluminescence assay. RESULTS: Glufosfamide induced cytotoxicity in HepG2 cells in a concentration- and time-dependent manner. The IC50 values for glufosfamide were significantly lower compared to ifosfamide. The frequency of apoptosis was much higher for glufosfamide than that of ifosfamide. The contents of caspase-9 and caspase-3 were elevated following exposure to GLU more than IFO. The anti-apoptotic Bcl2 gene expression, the mitochondrial membrane potential, and the cellular ATP levels were significantly decreased than in case of ifosfamide. CONCLUSIONS: The current study reported for the first time cytotoxicity activity of glufosfamide in HepG2 cells in vitro. The obtained results confirmed the higher oncolytic activity of glufosfamide than its aglycone ifosfamide. The generated data warrants further elucidations by in vivo study.

Fine-tuning ER-phagy by post-translational modifications.

Autophagy functions in both selective and non-selective ways to maintain cellular homeostasis. Endoplasmic reticulum autophagy (ER-phagy) is a subclass of autophagy responsible for the degradation of the endoplasmic reticulum through selective encapsulation into autophagosomes. ER-phagy occurs both under physiological conditions and in response to stress cues, and plays a crucial role in maintaining the homeostatic control of the organelle. Although specific receptors that target parts of the ER membrane, as well as, internal proteins for lysosomal degradation have been identified, the molecular regulation of ER-phagy has been elusive. Recent work has uncovered novel regulators of ER-phagy that involve post-translational modifications of ER-resident proteins and functional cross-talk with other cellular processes. Herein, we discuss how morphology affects the function of the peripheral ER, and how ER-phagy modulates the turnover of this organelle. We also address how ER-phagy is regulated at the molecular level, considering implications relevant to human diseases.

Studies on vascular response to full superantigens and superantigen derived peptides: Possible production of novel superantigen variants with less vasodilation effect for tolerable cancer immunotherapy.

Superantigens (SAgs) are a class of antigens that cause non-specific activation of T-cells resulting in polyclonal T cell activation and massive cytokine release and causing symptoms similar to sepsis, e.g. hypotension and subsequent hyporeactivity. We investigated the direct effect of SAgs on vascular tone using two recombinant SAgs, SEA and SPEA. The roles of Nitric Oxide (NO) and potentially hyperpolarization, which is dependent on the K+ channel activation, were also explored. The data show that SEA and SPEA have direct vasodilatory effects that were in part NO-dependent, but completely dependent on activation of K+ channels. Our work also identified the functional regions of one of the superantigens, SPEA, that are involved in causing the vasodilation and possible hypotension. A series of 20 overlapping peptides, spanning the entire sequence of SPEA, were designed and synthesized. The vascular response of each peptide was measured, and the active peptides were identified. Our results implicate the regions, (61-100), (101-140) and (181-220) which cause the vasodilation and possible hypotension effects of SPEA. The data also shows that the peptide 181-220 exert the highest vasodilation effect. This work therefore, demonstrates the direct effect of SAgs on vascular tone and identify the active region causing this vasodilation. We propose that these three peptides could be effective novel antihypertensive drugs. We also overexpressed, in E.coli, four superantigens from codon optimized genes.

Production of "biobetter" variants of glucarpidase with enhanced enzyme activity.

Glucarpidase, also known as carboxypeptidase G2, is a Food and Drug Administration-approved enzyme used in targeted cancer strategies such as antibody-directed enzyme prodrug therapy (ADEPT). It is also used in drug detoxification when cancer patients have excessive levels of the anti-cancer agent methotrexate. The application of glucarpidase is limited by its potential immunogenicity and limited catalytic efficiency. To overcome these pitfalls, mutagenesis was applied to the glucarpidase gene of Pseudomonas sp. strain RS-16 to isolate three novels "biobetter" variants with higher specific enzyme activity. DNA sequence analysis of the genes for the variants showed that each had a single point mutation, resulting in the amino acid substitutions: I100 T, G123S and T239 A. Km, Vmax and Kcat measurements confirmed that each variant had increased catalytic efficiency relative to wild type glucarpidase. Additionally, circular dichroism studies indicated that they had a higher alpha-helical content relative to the wild type enzyme. However, three different software packages predicted that they had reduced protein stability, which is consistent with having higher activities as a tradeoff. The novel glucarpidase variants presented in this work could pave the way for more efficient drug detoxification and might allow dose escalation during chemotherapy. They also have the potential to increase the efficiency of ADEPT and to reduce the number of treatment cycles, thereby reducing the risk that patients will develop antibodies to glucarpidase.

Production of "biobetter" glucarpidase variants to improve drug detoxification and antibody directed enzyme prodrug therapy for cancer treatment.

Recombinant glucarpidase (formerly: Carboxypeptidase G2, CPG2) is used in Antibody Directed Enzyme Prodrug Therapy (ADEPT) for the treatment of cancer. In common with many protein therapeutics, glucarpidase has a relatively short half-life in serum and, due to the need for the repeated cycles of the ADEPT, its bioavailability may be further diminished by neutralizing antibodies produced by patients. PEGylation and fusion with human serum albumin (HSA) are two approaches that are commonly employed to increase the residency time of protein therapeutics in blood, and also to increase the half-lives of the proteins in vivo. To address this stability and the immunogenicity problems, 'biobetter' glucarpidase variants, mono-PEGylated glucarpidase, and HSA fused glucarpidase by genetic fusion with albumin, were produced. Biochemical and bioactivity analyses, including anti-proliferation, bioassays, circular dichroism, and in vitro stability using human blood serum and immunoassays, demonstrated that the functional activities of the designed glucarpidase conjugates were maintained. The immunotoxicity studies indicated that the PEGylated glucarpidase did not significantly induce T-cell proliferation, suggesting that glucarpidase epitopes were masked by the PEG moiety. However, free glucarpidase and HSA-glucarpidase significantly increased T-cell proliferation compared with the negative control. In the latter case, this might be due to the type of expression system used or due to trace impurities associated with the highly purified (99.99%) recombinant HSA-glucarpidase. Both PEGylated glucarpidase and HAS-glucarpidase exhibit more stability in human serum and were more resistant to key human proteases relative to native glucarpidase. To our knowledge, this study is the first to report stable and less immunogenic glucarpidase variants produced by PEGylation and fusion with HSA. The results suggest that they may have better efficacy in drug detoxification and ADEPT, thereby improving this cancer treatment strategy.

Isolation and molecular characterization of novel glucarpidases: Enzymes to improve the antibody directed enzyme pro-drug therapy for cancer treatment.

Repeated cycles of antibody-directed enzyme pro-drug therapy (ADEPT) and the use of glucarpidase in the detoxification of cytotoxic methotrexate (MTX) are highly desirable during cancer therapy but are hampered by the induced human antibody response to glucarpidase. Novel variants of glucarpidase (formal name: carboxypeptidase G2, CPG2) with epitopes not recognized by the immune system are likely to allow repeated cycles of ADEPT for effective cancer therapy. Towards this aim, over two thousand soil samples were collected and screened for folate hydrolyzing bacteria using folate as the sole carbon source. The work led to the isolation and the characterization of three new glucarpidase producing strains, which were designated as: Pseudomonas lubricans strain SF168, Stenotrophomonas sp SA and Xenophilus azovorans SN213. The CPG2 genes of Xenophilus azovorans SN213 (named Xen CPG2) and Stenotrophomonas sp SA (named Sten CPG2) were cloned and molecularly characterized. Both Xen CPG2 and Sten CPG2 share very close amino acid sequences (99%); we therefore, focused on the study of Xen CPG2. Finally, we demonstrated that a polyclonal antibody raised against our new CPG2, Xen CPG2, does not react with the CPG2 from Pseudomonas sp. strain RS-16 (Ps CPG2) that are currently in clinical use. The two enzymes, therefore could potentially be used consecutively in the ADEPT protocol to minimize the effect of the human antibody response that hampers current treatment with Ps CPG2. The identified novel CPG2 in this study will, therefore, pave the way for safer antibody directed enzyme pro-drug therapy for cancer treatment.