A novel cell biological tool to explain mechanics and dynamics in fission yeast.

The Rho guanosine triphosphatase hydrolase enzyme (GTPase) is required for the control of the actin cytoskeleton, but its activation in vivo condition is unknown. The study's goal was to find a new synthetic nanobody VHH (P-36 tagged with mNeonGreen) that interacts strongly with the Rho GTPase. We present the first novel synthetic nanobody, VHH (P-36 tagged with mNeonGreen), tested in fission yeast cells and found to have a particular interaction with Rho1GTPase. Plasmids were constructed by using of certain enzymes to digest the pDUAL-pef1a vector plasmid to produce a protein that was encoded by cloned genes. A varied VHH library was created synthetically, then transformed into yeast cells, and positive clones were chosen using chemical agents. To investigate protein interactions and cellular reactions, several studies were carried out, such as live cell imaging, growth curve analysis, coimmunoprecipitation, structural analysis, and cell therapies. Prism and RStudio were used for the statistical analysis. The presence of VHH (P-36) has no effect on the growth pattern making it an appropriate model for studying cytokinesis in vivo. According to a computational biological study, its affinity to interact with Rho1GTPase with all the complementarity-determining region (CDR) regions found on VHH (P-36) is extremely strong. We were able to track its subcellular target by localization using a fluorescent confocal microscope, ensuring the maintenance of cell polarity and morphology. Spheroplast analysis revealed a circular-shaped cell with an even distribution of Rho1 tagged VHH (P-36), indicating that the interaction occurs near the plasma membrane. The introduction of latrunculin-A (Lat-A) disrupted Rho GTPase localization, demonstrating the control over actin production, and the cell did not show evidence of mitotic phase commencement while Lat-A was present. Finally, this important biological tool can aid in our understanding of the mechanics and dynamics of cytokinesis in relation to Rho1GTPase.

Mitigating a Bioactivation Liability with an Azetidine-Based Inhibitor of Diacylglycerol Acyltransferase 2 (DGAT2) En Route to the Discovery of the Clinical Candidate Ervogastat.

We recently disclosed SAR studies on systemically acting, amide-based inhibitors of diacylglycerol acyltransferase 2 (DGAT2) that addressed metabolic liabilities with the liver-targeted DGAT2 inhibitor PF-06427878. Despite strategic placement of a nitrogen atom in the dialkoxyaromatic ring in PF-06427878 to evade oxidative O-dearylation, metabolic intrinsic clearance remained high due to extensive piperidine ring oxidation as exemplified with compound 1. Piperidine ring modifications through alternate N-linked heterocyclic ring/spacer combination led to azetidine 2 that demonstrated lower intrinsic clearance. However, 2 underwent a facile cytochrome P450 (CYP)-mediated α-carbon oxidation followed by azetidine ring scission, resulting in the formation of ketone (M2) and aldehyde (M6) as stable metabolites in NADPH-supplemented human liver microsomes. Inclusion of GSH or semicarbazide in microsomal incubations led to the formation of Cys-Gly-thiazolidine (M3), Cys-thiazolidine (M5), and semicarbazone (M7) conjugates, which were derived from reaction of the nucleophilic trapping agents with aldehyde M6. Metabolites M2 and M5 were biosynthesized from NADPH- and l-cysteine-fortified human liver microsomal incubations with 2, and proposed metabolite structures were verified using one- and two-dimensional NMR spectroscopy. Replacement of the azetidine substituent with a pyridine ring furnished 8, which mitigated the formation of the electrophilic aldehyde metabolite, and was a more potent DGAT2 inhibitor than 2. Further structural refinements in 8, specifically introducing amide bond substituents with greater metabolic stability, led to the discovery of PF-06865571 (ervogastat) that is currently in phase 2 clinical trials for the treatment of nonalcoholic steatohepatitis.

Toxic effects of AZD1208 on mouse oocytes and its possible mechanisms.

AZD1208, a pan-inhibitor that can effectively inhibit PIM kinase, is used for the treatment of advanced solid tumors and malignant lymphomas. Numerous studies have proved its curative effects while its potential cellular toxicity on reproduction was still little known. In this study, we investigated the toxic effects of AZD1208 on mouse oocytes. The results showed that AZD1208 treatment did not affect meiotic resumption, but postponed oocyte maturation as indicated by delayed first polar body extrusion. Further mechanistic study showed that AZD1208 treatment delayed spindle assembly. In addition, we found that oocytes treated with AZD1208 showed mitochondrial dysfunction. Abnormal mitochondrial clusters with decreased mitochondrial membrane potential were observed in oocytes during incubation in vitro. Moreover, increased oxidative stress was observed by testing the level of reactive oxygen species. In summary, our results suggest that AZD1208 treatment influences oocyte meiotic progression by causing mitochondrial dysfunctions and subsequent delayed spindle assembly.

Bicyclic Picomolar OGA Inhibitors Enable Chemoproteomic Mapping of Its Endogenous Post-translational Modifications.

Owing to its roles in human health and disease, the modification of nuclear, cytoplasmic, and mitochondrial proteins with O-linked N-acetylglucosamine residues (O-GlcNAc) has emerged as a topic of great interest. Despite the presence of O-GlcNAc on hundreds of proteins within cells, only two enzymes regulate this modification. One of these enzymes is O-GlcNAcase (OGA), a dimeric glycoside hydrolase that has a deep active site cleft in which diverse substrates are accommodated. Chemical tools to control OGA are emerging as essential resources for helping to decode the biochemical and cellular functions of the O-GlcNAc pathway. Here we describe rationally designed bicyclic thiazolidine inhibitors that exhibit superb selectivity and picomolar inhibition of human OGA. Structures of these inhibitors in complex with human OGA reveal the basis for their exceptional potency and show that they extend out of the enzyme active site cleft. Leveraging this structure, we create a high affinity chemoproteomic probe that enables simple one-step purification of endogenous OGA from brain and targeted proteomic mapping of its post-translational modifications. These data uncover a range of new modifications, including some that are less-known, such as O-ubiquitination and N-formylation. We expect that these inhibitors and chemoproteomics probes will prove useful as fundamental tools to decipher the mechanisms by which OGA is regulated and directed to its diverse cellular substrates. Moreover, the inhibitors and structures described here lay out a blueprint that will enable the creation of chemical probes and tools to interrogate OGA and other carbohydrate active enzymes.

Computational and Structure-Based Development of High Potent Cell-Active Covalent Inhibitor Targeting the Peptidyl-Prolyl Isomerase NIMA-Interacting-1 (Pin1).

The unique proline isomerase peptidyl-prolyl isomerase NIMA-interacting-1 (Pin1) is reported to activate numerous cancer-driving pathways simultaneously, and aberrant Pin1 activation is present in many human cancers. Here, we identified a novel hit compound, ZL-Pin01, that covalently modified Pin1 at Cys113 with an half-maximal inhibitory concentration (IC50) of 1.33 ± 0.07 µM through screening an in-house library. Crystallographic study drove the process of structure-guided optimization and led to the potent inhibitor ZL-Pin13 with an IC50 of 0.067 ± 0.03 µM. We obtained four co-crystal structures of Pin1 complexed with inhibitors that elucidated the detailed binding mode of the derivatives with Pin1. Interestingly, the co-crystal of Pin1 with ZL-Pin13 obtained by co-crystallization revealed the conformational change of Gln129 induced by the inhibitor. Furthermore, ZL-Pin13 effectively inhibited the proliferation and downregulated the Pin1 substrates in MDA-MB-231 cells. Collectively, we developed a potent covalent inhibitor of Pin1, ZL-Pin13, which could be an effective probe for studying the functional roles of Pin1.

Evidence for Proline Catabolic Enzymes in the Metabolism of Thiazolidine Carboxylates.

Thiazolidine carboxylates such as thiazolidine-4-carboxylate (T4C) and thiazolidine-2-carboxylate (T2C) are naturally occurring sulfur analogues of proline. These compounds have been observed to have both beneficial and toxic effects in cells. Given that proline dehydrogenase has been proposed to be a key enzyme in the oxidative metabolism of thioprolines, we characterized T4C and T2C as substrates of proline catabolic enzymes using proline utilization A (PutA), which is a bifunctional enzyme with proline dehydrogenase (PRODH) and l-glutamate-γ-semialdehyde dehydrogenase (GSALDH) activities. PutA is shown here to catalyze the FAD-dependent PRODH oxidation of both T4C and T2C with catalytic efficiencies significantly higher than with proline. Stopped-flow experiments also demonstrate that l-T4C and l-T2C reduce PutA-bound FAD at rates faster than proline. Unlike proline, however, oxidation of T4C and T2C does not generate a substrate for NAD+-dependent GSALDH. Instead, PutA/PRODH oxidation of T4C leads to cysteine formation, whereas oxidation of T2C generates an apparently stable Δ4-thiazoline-2-carboxylate species. Our results provide new insights into the metabolism of T2C and T4C.

Kinetics of human pyrroline-5-carboxylate reductase in L-thioproline metabolism.

L-Thioproline (L-thiazolidine-4-carboxylate, L-T4C) is a cyclic sulfur-containing analog of L-proline found in multiple kingdoms of life. The oxidation of L-T4C leads to L-cysteine formation in bacteria, plants, mammals, and protozoa. The conversion of L-T4C to L-Cys in bacterial cell lysates has been attributed to proline dehydrogenase and L-Δ1-pyrroline-5-carboxylate (P5C) reductase (PYCR) enzymes but detailed kinetic studies have not been conducted. Here, we characterize the dehydrogenase activity of human PYCR isozymes 1 and 2 with L-T4C using NAD(P)+ as the hydride acceptor. Both PYCRs exhibit significant L-T4C dehydrogenase activity; however, PYCR2 displays nearly tenfold higher catalytic efficiency (136 M-1 s-1) than PYCR1 (13.7 M-1 s-1). Interestingly, no activity was observed with either L-Pro or the analog DL-thiazolidine-2-carboxylate, indicating that the sulfur at the 4-position is critical for PYCRs to utilize L-T4C as a substrate. Inhibition kinetics show that L-Pro is a competitive inhibitor of PYCR1 [Formula: see text] with respect to L-T4C, consistent with these ligands occupying the same binding site. We also confirm by mass spectrometry that L-T4C oxidation by PYCRs leads to cysteine product formation. Our results suggest a new enzyme function for human PYCRs in the metabolism of L-T4C.

Surface-Modified Colloid CdTe/CdS Quantum Dots by a Biocompatible Thiazolidine Derivative as Promising Platform for Immobilization of Glucose Oxidase: Application to Fluorescence Sensing of Glucose.

This work focuses on the synthesis of novel modified core-shell CdTe/CdS quantum dots (QDs) and develops as a fluorescence sensor for glucose determination. The (E)-2,2'-(4,4'-dioxo-2,2'-dithioxo-2H,2'H-[5,5'-bithiazolylidene]-3,3'(4H,4'H)-diyl)bis(3- mercaptopropanoic acid) (DTM) as a new derivative of thiazolidine was synthesized and characterized and used to surface-modification of CdTe/CdS QDs. DTM-capped CdTe/CdS QDs used to immobilization of glucose oxidase (GOD). The intensity fluorescence emission of the CdSe/CdS-DTM/GOD is highly sensitive to the concentration of H2O2 as a byproduct of the catalytic oxidation of glucose. The experimental results showed that the quenched fluorescence was proportional to the glucose concentration within the range of 10 nM-0.32 µM under optimized experimental conditions. The limit of detection of this system was found to be 4.3 nM. Compared with most of the existing methods, this newly developed system possesses many advantages, including simplicity, low cost, and good sensitivity.

Discovery of small molecule acting as multitarget inhibitor of colorectal cancer by simultaneous blocking of the key COX-2, 5-LOX and PIM-1 kinase enzymes.

Colorectal cancer (CRC) is the second cause of cancer death worldwide. Inhibitors of COX-2, 5-LOX and PIM-1 kinase were very effective in the treatment and prevention of CRC in mouse models in vivo. Furthermore, thymol was confirmed to inhibit CRC cell proliferation in cancer cell lines and inhibitory activity against COX-2 and 5-LOX. On the other hand, 4-thiazolidinone pharmacophore was incorporated in the structures of various reported COX-2, 5-LOX and PIM kinase inhibitors. Consequently, the aim of the present investigation was to combat CRC by synthesis and biological evaluation of new thymol - 4-thiazolidinone hybrids as multitarget anticancer agents that could inhibit the key COX-2, 5-LOX and PIM-1 kinase enzymes simultaneously. Compounds 5a-d and 5g displayed inhibitory activity against COX-2 nearly equal to Celecoxib with high selectivity index (SI). Moreover, compounds 5b-e showed 5-LOX inhibitory activity nearly equal to the reference Quercetin while compounds 5a, 5f and 5g elicited inhibitory activity slightly lower than Quercetin. Furthermore, in vivo formalin-induced paw edema test revealed that, compounds 5a, 5c, 5f and 5g showed higher % inhibition than Celecoxib and compounds 5a, 5f and 5g showed higher % inhibition than Diclofenac sodium. In addition, compounds 5a-c, 5e-g showed in vivo superior gastrointestinal safety profile as Celecoxib in fasted rats. Besides, compounds 5d, 5e and 5g exhibited the highest activity against human CRC cell lines (Caco-2 and HCT-116) at doses less than their EC100 on normal human cells. Furthermore, compounds 5e and 5g induced apoptosis-dependent death by above 50% in the treated CRC cell lines. Moreover, compounds 5e and 5g induced caspase activation by >50% in human CRC. Also, compounds 5d, 5e and 5g showed in vitro inhibitory activity against both PIM-1\2 kinases comparable to the reference Staurosporine. In silico docking studies were concordant with the biological results. In conclusion, compound 5g, of simple chemical structure, achieved the target goal of inhibiting three targets leading to inhibition of human CRC cell proliferation. It inhibited the target key enzymes COX-2, 5-LOX and PIM-1\2 kinase in vitro. Besides, it revealed in vitro inhibition of cell proliferation in cancer cell lines via activation of caspase 3\7 dependent-apoptosis in human CRC cell lines. In addition, it elicited in vivo anti-inflammatory activity in formalin-induced paw edema test and in vivo oral safety in gastric ulcerogenic activity test.

Synthesis and analysis of 4-oxothiazolidines as potential dual inhibitors of deoxyribonuclease I and xanthine oxidase.

In this study, seven new 4-oxothiazolidine derivatives were synthesized and assayed, along 7 known derivatives, for inhibitory properties against deoxyribonuclease I (DNase I) and xanthine oxidase (XO) in vitro. Among tested compounds, (5Z)-Ethyl-2-(2-(cyanomethylene)-4-oxothiazolidin-5-yliden)acetate (6) exhibited inhibitory activity against both enzymes (DNase I IC50 = 67.94 ± 5.99 µM; XO IC50 = 98.98 ± 13.47 µM), therefore being the first reported dual inhibitor of DNase I and XO. Observed DNase I inhibition qualifies compound 6 as the most potent small organic DNase I inhibitor reported so far. Derivatives of 2-alkyliden-4-oxothiazolidinone (1) inhibited DNase I below 200 µM, while the other tested 4-oxothiazolidine derivatives remained inactive against both enzymes. The molecular docking and molecular dynamics simulations into the binding sites of DNase I and XO enzyme allowed us to clarify the binding modes of this 4-oxothiazolidine derivative, which might aid future development of dual DNase I and XO.

2-Aryl-3-(6-trifluoromethoxy)benzo[d]thiazole-based thiazolidinone hybrids as potential anti-infective agents: Synthesis, biological evaluation and molecular docking studies.

The search for new antimicrobial agents is greater than ever due to the perpetual threat of multidrug resistance in known pathogens and the relentless emergence of new infections. In this manuscript, ten thiazole-based thiazolidinone hybrids bearing a 6-trifluoromethoxy substituent on the benzothiazole core were synthesized and evaluated against a panel of four bacterial strains Salmonella typhimurium, Staphylococcus aureus, Escherichia coli and Listeria monocytogenes and three resistant strains Pseudomonas aeruginosa, E. coli and MRSA. The evaluation of minimum bactericidal and minimum inhibitory concentrations was accomplished by microdilution assay. As reference compounds ampicillin and streptomycin were employed. All compounds displayed antibacterial efficiencies with MBCs/MICs at 0.25-1 mg/mL and 0.12-1 mg/mL respectively while ampicillin displayed MBCs/MICs at 0.15-0.3 mg/mL and at 0.1-0.2 mg/mL respectively. MICs/MBC of streptomycin varied from 0.05 to 0.15 mg/mL and from 0.1 to 0.3 mg/mL respectively. The best overall effect was observed for compound h4, while compound h1 exhibited the highest effective action against E. coli (MIC/MBC 0.12/0.25 mg/ml) among all tested compounds.

Synthesis of metronidazole based thiazolidinone analogs as promising antiamoebic agents.

Metronidazole and its derivatives are widely used for the treatment of amoebiasis. However, metronidazole is considered as the standard drug but it has many side effects. The present study describes the synthesis of a series of metronidazole based thiazolidinone analogs via Knoevenagel condensation of 4-[2-(2-methyl-5-nitro-1H-imidazole-1-yl)ethoxy]benzaldehyde 1 with various thiazolidinone derivatives 2-14 to get the new scaffold (15-27) having better activity and lesser toxicity. Six compounds have shown better efficacy and lesser cytotoxicity than the standard drug metronidazole towards HM1: IMSS strain of Entamoeba histolytica. These compounds may combat the problem of drug resistance and might be effective in identifying potential alternatives for future drug discovery against EhOASS.

A novel quantification method for sulfur-containing biomarkers of formaldehyde and acetaldehyde exposure in human urine and plasma samples.

A novel method for the quantification of the sulfur-containing metabolites of formaldehyde (thiazolidine carboxylic acid (TCA) and thiazolidine carbonyl glycine (TCG)) and acetaldehyde (methyl thiazolidine carboxylic acid (MTCA) and methyl thiazolidine carbonyl glycine (MTCG)) was developed and validated for human urine and plasma samples. Targeting the sulfur-containing metabolites of formaldehyde and acetaldehyde in contrast to the commonly used biomarkers formate and acetate overcomes the high intra- and inter-individual variance. Due to their involvement in various endogenous processes, formate and acetate lack the required specificity for assessing the exposure to formaldehyde and acetaldehyde, respectively. Validation was successfully performed according to FDA's Guideline for Bioanalytical Method Validation (2018), showing excellent performance with regard to accuracy, precision, and limits of quantification (LLOQ). TCA, TCG, and MTCG proved to be stable under all investigated conditions, whereas MTCA showed a depletion after 21 months. The method was applied to a set of pilot samples derived from smokers who consumed unfiltered cigarettes spiked with 13C-labeled propylene glycol and 13C-labeled glycerol. These compounds were used as potential precursors for the formation of 13C-formaldehyde and 13C-acetaldehyde during combustion. Plasma concentrations were significantly lower as compared to urine, suggesting urine as suitable matrix for a biomonitoring. A smoking-related increase of unlabeled biomarker concentrations could not be shown due to the ubiquitous distribution in the environment. While the metabolites of 13C-acetaldehyde were not detected, the described method allowed for the quantification of 13C-formaldehyde uptake from cigarette smoking by targeting the biomarkers 13C-TCA and 13C-TCG in urine.Graphical abstract.

LC-MS/MS Coupled with a Stable-Isotope Dilution Method for the Quantitation of Thioproline-Glycine: A Novel Metabolite in Formaldehyde- and Oxidative Stress-Exposed Cells.

Formaldehyde (FA) is a human carcinogen that is ubiquitous in the ambient environment and also generated endogenously in oxidatively stressed cells. There is accumulated evidence that FA is an etiological agent of leukemia development in humans. To develop a biomarker for FA exposure, we have, in this study, developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) coupled with stable isotope-dilution method to explore the reactivity of FA with glutathione (GSH) in physiologically relevant conditions. Interestingly, analysis of the reaction mixture by LC-MS/MS revealed exposure concentration- and duration-dependent formation of thioproline-glycine (SPro-Gly) produced by reaction of FA with cysteinyl-glycine (Cys-Gly) as a novel metabolite. Furthermore, dose-dependent formation of the thioproline adduct was observed in human cells separately exposed to FA and Fe2+-EDTA, a hydroxyl radical source. To the best of our knowledge, this is the first study reporting a thiazolidine carboxylic acid formed by reaction of FA and Cys-Gly is a major metabolite of FA. The results suggest a variety of GSH-derived thiazolidine metabolites may serve as potential biomarkers for FA and oxidative stress exposure, and the developed LC-MS/MS method provides a means for accurate determination of SPro-Gly as a dosimeter of oxidative stress and formaldehyde exposure.

2-(3-Hydroxy-5-phosphonooxymethyl-2-methyl-4-pyridyl)-1,3-thiazolidine-4-carboxylic Acid, Novel Metabolite of Pyridoxal 5'-Phosphate and Cysteine Is Present in Human Plasma-Chromatographic Investigations.

It is well-established that aminothiols, to which cysteine (Cys) belongs, are highly reactive towards aldehydes in an aqueous environment, forming substituted thiazolidine carboxylic acids. This report provides evidence that formation of the product containing a thiazolidine ring through non-enzymatic condensation of Cys and an active form of vitamin B6 pyridoxal 5'-phosphate (PLP) occurs in vivo in humans. To prove this point, a new method, based on a gas chromatography coupled with mass spectrometry (GC-MS), has been designed to identify and quantify Cys and PLP adduct, 2-(3-hydroxy-5-phosphonooxymethyl-2-methyl-4-pyridyl)-1,3-thiazolidine-4-carboxylic acid (HPPTCA) in human plasma. The GC-MS assay relies on sample deproteinization by ultrafiltration over cut-off membranes and preconcentration by drying under vacuum, followed by treatment of the residue with derivatization mixture containing anhydrous pyridine, N-trimethylsilyl-N-methyl trifluoroacetamide (MSTFA) and trimethylchlorosilane (TMCS). The method quantifies HPPTCA in a linear range from 1 to 20 µmol L-1, where the lowest standard on the calibration curve refers to the limit of quantification (LOQ). The validity of the method was demonstrated. Furthermore, the method was successfully applied to plasma samples donated by apparently healthy volunteers and breast cancer patients. The GC-MS assay provides a new tool that will hopefully facilitate studies on the role of HPPTCA in living systems.

Synthesis of (4R)-thiazolidine carboxylic acid and evaluation of antioxidant potential.

We report here the synthesis as well as antioxidant activity of a series of 2-aryl thiazolidine-4-carboxylic acids, including two novel derivatives. They were synthesized by nucleophilic cyclic condensation of L-cysteine hydrochloride with a range of aromatic aldehydes. Their in vitro antioxidant activity was evaluated by DPPH radical scavenging assay. It was observed that the aromatic substituent at C-2 of thiazolidine ring effects the antioxidant potential of the thiazolidine derivatives. The nature and position of the substituents on aromatic ring were correlated with antioxidant activity. Compounds with -OCH3 group on aromatic ring showed a better radical scavenging property than the other groups such as -Cl, -F, and -NO2. The presence of phenyl ring thus enhanced radical scavenging activity.

Discovery and Structure-Activity Relationship Study of (Z)-5-Methylenethiazolidin-4-one Derivatives as Potent and Selective Pan-phosphatidylinositol 5-Phosphate 4-Kinase Inhibitors.

Due to their role in many important signaling pathways, phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) are attractive targets for the development of experimental therapeutics for cancer, metabolic, and immunological disorders. Recent efforts to develop small molecule inhibitors for these lipid kinases resulted in compounds with low- to sub-micromolar potencies. Here, we report the identification of CVM-05-002 using a high-throughput screen of PI5P4Kα against our in-house kinase inhibitor library. CVM-05-002 is a potent and selective inhibitor of PI5P4Ks, and a 1.7 Å X-ray structure reveals its binding interactions in the ATP-binding pocket. Further investigation of the structure-activity relationship led to the development of compound 13, replacing the rhodanine-like moiety present in CVM-05-002 with an indole, a potent pan-PI5P4K inhibitor with excellent kinome-wide selectivity. Finally, we employed isothermal cellular thermal shift assays (CETSAs) to demonstrate the effective cellular target engagement of PI5P4Kα and -ß by the inhibitors in HEK 293T cells.

Thioproline formation as a driver of formaldehyde toxicity in Escherichia coli.

Formaldehyde (HCHO) is a reactive carbonyl compound that formylates and cross-links proteins, DNA, and small molecules. It is of specific concern as a toxic intermediate in the design of engineered pathways involving methanol oxidation or formate reduction. The interest in engineering these pathways is not, however, matched by engineering-relevant information on precisely why HCHO is toxic or on what damage-control mechanisms cells deploy to manage HCHO toxicity. The only well-defined mechanism for managing HCHO toxicity is formaldehyde dehydrogenase-mediated oxidation to formate, which is counterproductive if HCHO is a desired pathway intermediate. We therefore sought alternative HCHO damage-control mechanisms via comparative genomic analysis. This analysis associated homologs of the Escherichia coli pepP gene with HCHO-related one-carbon metabolism. Furthermore, deleting pepP increased the sensitivity of E. coli to supplied HCHO but not other carbonyl compounds. PepP is a proline aminopeptidase that cleaves peptides of the general formula X-Pro-Y, yielding X + Pro-Y. HCHO is known to react spontaneously with cysteine to form the close proline analog thioproline (thiazolidine-4-carboxylate), which is incorporated into proteins and hence into proteolytic peptides. We therefore hypothesized that certain thioproline-containing peptides are toxic and that PepP cleaves these aberrant peptides. Supporting this hypothesis, PepP cleaved the model peptide Ala-thioproline-Ala as efficiently as Ala-Pro-Ala in vitro and in vivo, and deleting pepP increased sensitivity to supplied thioproline. Our data thus (i) provide biochemical genetic evidence that thioproline formation contributes substantially to HCHO toxicity and (ii) make PepP a candidate damage-control enzyme for engineered pathways having HCHO as an intermediate.

Induction of Cryptic Antifungal Pulicatin Derivatives from Pantoea agglomerans by Microbial Co-Culture.

Microbial co-culture or mixed fermentation proved to be an efficient strategy to expand chemical diversity by the induction of cryptic biosynthetic pathways, and in many cases led to the production of new antimicrobial agents. In the current study, we report a rare example of the induction of silent/cryptic bacterial biosynthetic pathway by the co-culture of Durum wheat plant roots-associated bacterium Pantoea aggolomerans and date palm leaves-derived fungus Penicillium citrinum. The initial co-culture indicated a clear fungal growth inhibition which was confirmed by the promising antifungal activity of the co-culture total extract against Pc. LC-HRMS chemical profiling demonstrated a huge suppression in the production of secondary metabolites (SMs) of axenic cultures of both species with the emergence of new metabolites which were dereplicated as a series of siderophores. Large-scale co-culture fermentation led to the isolation of two new pulicatin derivatives together with six known metabolites which were characterised using HRESIMS and NMR analyses. During the in vitro antimicrobial evaluation of the isolated compounds, pulicatin H (2) exhibited the strongest antifungal activity against Pc, followed by aeruginaldehyde (1) and pulicatin F (4), hence explaining the initial growth suppression of Pc in the co-culture environment.

Review on Chemistry, Analysis and Pharmacology of Teneligliptin: A Novel DPP-4 Inhibitor.

This article provides comprehensive and collective facts about teneligliptin. Teneligliptin is a dipeptide peptidase-4 (DPP-4) inhibitor that belongs to the third generation, used in the management of type 2 diabetes. It inhibits human DPP-4 enzyme activity. This drug falls under class 3; it interacts with S1, S2, and S2E extensive sub-sites. Teneligliptin and its metabolites are mainly determined in the human plasma matrix by hyphenated chromatographic methods. These developed methods could be foreseen for their clinical applications. Moreover, the stress degradation studies of Teneligliptin under different stress conditions provide an insight into degradation pathways and help in the elucidation of the structure of the degradation products by liquid mass spectroscopy. These methods are also used for routine quality control analysis of teneligliptin in pharmaceutical dosage forms.