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Protein Engineering, Design & Selection... Feb 2022Proline dehydrogenase (PRODH) catalyzes the FAD-dependent oxidation of l-proline to Δ1-pyrroline-5-carboxylate and is a target for inhibitor discovery because of its...
Proline dehydrogenase (PRODH) catalyzes the FAD-dependent oxidation of l-proline to Δ1-pyrroline-5-carboxylate and is a target for inhibitor discovery because of its importance in cancer cell metabolism. Because human PRODH is challenging to purify, the PRODH domains of the bacterial bifunctional enzyme proline utilization A (PutA) have been used for inhibitor development. These systems have limitations due to large polypeptide chain length, conformational flexibility and the presence of domains unrelated to PRODH activity. Herein, we report the engineering of minimal PRODH domains for inhibitor discovery. The best designs contain one-third of the 1233-residue PutA from Sinorhizobium meliloti and include a linker that replaces the PutA α-domain. The minimal PRODHs exhibit near wild-type enzymatic activity and are susceptible to known inhibitors and inactivators. Crystal structures of minimal PRODHs inhibited by S-(-)-tetrahydro-2-furoic acid and 2-(furan-2-yl)acetic acid were determined at 1.23 and 1.72 Å resolution. Minimal PRODHs should be useful in chemical probe discovery.
Topics: Humans; Proline Oxidase; Proline; Bacterial Proteins
PubMed: 36448708
DOI: 10.1093/protein/gzac016 -
Drugs Sep 2018Trigeminal neuralgia is a representative neuropathic facial pain condition, characterised by unilateral paroxysmal pain in the distribution territory of one or more... (Review)
Review
Trigeminal neuralgia is a representative neuropathic facial pain condition, characterised by unilateral paroxysmal pain in the distribution territory of one or more divisions of the trigeminal nerve, triggered by innocuous stimuli. A subgroup of patients with trigeminal neuralgia [TN (previously defined as atypical TN)] also suffer from concomitant continuous pain, i.e. a background pain between the paroxysmal attacks. The aim of this review is to provide current, evidence-based, knowledge about the pharmacological treatment of typical and atypical TN, with a specific focus on drugs in development. We searched for relevant papers within PubMed, EMBASE, the Cochrane Database of Systematic Reviews and the Clinical Trials database (ClinicalTrials.gov), taking into account publications up to February 2018. Two authors independently selected studies for inclusions, data extraction, and bias assessment. Carbamazepine and oxcarbazepine are the first-choice drugs for paroxysmal pain. When sodium channel blockers cannot reach full dosage because of side effects, an add-on treatment with lamotrigine or baclofen should be considered. In patients with atypical TN, both gabapentin and antidepressants are expected to be efficacious and should be tried as an add-on to oxcarbazepine or carbamazepine. Although carbamazepine and oxcarbazepine are effective in virtually the totality of patients, they are responsible for side effects causing withdrawal from treatment in an important percentage of cases. A new, better tolerated, Nav1.7 selective state-dependent, sodium channel blocker (vixotrigine) is under development. Future trials testing the effect of combination therapy in patients with TN are needed, especially in patients with concomitant continuous pain and in TN secondary to multiple sclerosis.
Topics: Anticonvulsants; Antidepressive Agents; Baclofen; Carbamazepine; Drug Therapy, Combination; Gabapentin; Humans; Lamotrigine; Oxcarbazepine; Phenyl Ethers; Proline; Sodium Channel Blockers; Trigeminal Neuralgia
PubMed: 30178160
DOI: 10.1007/s40265-018-0964-9 -
The Journal of Physical Chemistry. B Dec 2022The amino group of proline is part of a pyrrolidine ring, which makes it unique among the proteinogenic amino acids. To unravel its full electronic structure, proline in...
The amino group of proline is part of a pyrrolidine ring, which makes it unique among the proteinogenic amino acids. To unravel its full electronic structure, proline in solid state and aqueous solution is investigated using X-ray emission spectroscopy and resonant inelastic soft X-ray scattering. By controlling the pH value of the solution, proline is studied in its cationic, zwitterionic, and anionic configurations. The spectra are analyzed within a "building-block principle" by comparing with suitable reference molecules, i.e., acetic acid, cysteine, and pyrrolidine, as well as with spectral calculations based on density functional theory. We find that the electronic structure of the carboxyl group of proline is very similar to that of other amino acids as well as acetic acid. In contrast, the electronic structure of the amino group is significantly different and strongly influenced by the ring structure of proline.
Topics: X-Rays; Proline; Spectrometry, X-Ray Emission; Acetates
PubMed: 36418225
DOI: 10.1021/acs.jpcb.2c06557 -
Molecules (Basel, Switzerland) Nov 2021Orbitides are plant-derived small cyclic peptides with a wide range of biological activities. Phytochemical investigation of the whole plants of was performed with the...
Orbitides are plant-derived small cyclic peptides with a wide range of biological activities. Phytochemical investigation of the whole plants of was performed with the aim to discover new bioactive orbitides. Five undescribed proline-containing orbitides, dianthiamides A-E (-), were isolated from a methanolic extract of . Their structures were elucidated by extensive analysis of 1D and 2D NMR and HRESI-TOF-MS as well as ESI-MS/MS fragmentation data. The absolute configuration of the amino acid residues of compounds - was determined by Marfey's method. All compounds were tested for their cytotoxic activity, and dianthiamide A () exhibited weak activity against A549 cell line with IC value of 47.9 μM.
Topics: Amides; Carbon-13 Magnetic Resonance Spectroscopy; Cell Line, Tumor; Dianthus; Humans; Isomerism; Peptides, Cyclic; Proline; Proton Magnetic Resonance Spectroscopy
PubMed: 34885850
DOI: 10.3390/molecules26237275 -
Molecules (Basel, Switzerland) Oct 2020We designed and synthesized helical short oligopeptides with an L-proline on the N-terminus and hydrocarbon stapling on the side chain. Side-chain stapling is a...
We designed and synthesized helical short oligopeptides with an L-proline on the N-terminus and hydrocarbon stapling on the side chain. Side-chain stapling is a frequently used method for the development of biologically active peptides. Side-chain stapling can stabilize the secondary structures of peptides, and, therefore, stapled peptides may be applicable to peptide-based organocatalysts. Olefin-tethered -4-hydroxy-L-proline and L-serine and , and ()-α-allyl-proline were used as cross-linking motifs and incorporated into helical peptide sequences. The - and -selectivities were observed for the ring-closing metathesis reactions of peptides and (,+1 series), respectively, while no /-selectivity was observed for that of (,+3 series). The stapled peptide catalyzed the Michael addition reaction of 1-methylindole to α,β-unsaturated aldehyde, which was seven times faster than that of unstapled peptide . Furthermore, the high catalytic activity was retained even at lower catalyst loadings (5 mol %) and lower temperatures (0 °C). The circular dichroism spectra of stapled peptide showed a right-handed helix with a higher intensity than that of unstapled peptide . These results indicate that the introduction of side-chain stapling is beneficial for enhancing the catalytic activity of short oligopeptide catalysts.
Topics: Alkenes; Catalysis; Circular Dichroism; Hydrocarbons; Indoles; Oligopeptides; Proline; Protein Conformation; Protein Engineering; Structure-Activity Relationship
PubMed: 33066194
DOI: 10.3390/molecules25204667 -
Scientific Reports Jul 2023Capparis spinosa L. (caper) is a halophytic plant that grows in semi-arid or arid environments. The current study used an integrated experimental and computational...
Capparis spinosa L. (caper) is a halophytic plant that grows in semi-arid or arid environments. The current study used an integrated experimental and computational approach to investigate the network of inter-correlated effective variables on the activity of antioxidant enzymes, proline, and photosynthetic pigments in stressed caper. To investigate the possible relationships among intercorrelated variables and understand the possible mechanisms, predictive regression modelling, principal component analysis (PCA), Pearson's correlation, and path analysis were implemented. PCA successfully discerned different salt ratio- and drought-specific effects in data in the current study, and treatments with higher growth indices are easily recognizable. Different salt ratios did not have a significant effect on the activity of four antioxidant enzymes, proline and photosynthesis pigments content of caper. While at the mean level, the activity of four antioxidant enzymes of SOD, POD, CAT, and APX significantly increased under drought stress by 54.0%, 71.2%, 79.4%, and 117.6%, respectively, compared to 100% FC. The drought stress also significantly increased the content of carotemoid (29.3%) and proline (by 117.7%). Predictive equation models with highly significant R were developed for the estimation of antioxidant enzyme activity and proline content (> 0.94) as well as pigments (> 0.58) were developed. Path analysis studies revealed that proline is the most important regressor in four antioxidant enzyme activities, while leaf tissue density was the most effective variable in the case of chlorophylls. Furthermore, the network of intercorrelated variables demonstrated a close relationship between caper's antioxidant defence system, pigments, and morphological parameters under stress conditions. The findings of this study will be a useful guide to caper producers as well as plant ecophysiological researchers.
Topics: Antioxidants; Capparis; Photosynthesis; Chlorophyll; Proline
PubMed: 37524793
DOI: 10.1038/s41598-023-39683-5 -
Journal of the American Chemical Society Jul 2014The synthetic modification of proteins plays an important role in chemical biology and biomaterials science. These fields provide a constant need for chemical tools that...
The synthetic modification of proteins plays an important role in chemical biology and biomaterials science. These fields provide a constant need for chemical tools that can introduce new functionality in specific locations on protein surfaces. In this work, an oxidative strategy is demonstrated for the efficient modification of N-terminal residues on peptides and N-terminal proline residues on proteins. The strategy uses o-aminophenols or o-catechols that are oxidized to active coupling species in situ using potassium ferricyanide. Peptide screening results have revealed that many N-terminal amino acids can participate in this reaction, and that proline residues are particularly reactive. When applied to protein substrates, the reaction shows a stronger requirement for the proline group. Key advantages of the reaction include its fast second-order kinetics and ability to achieve site-selective modification in a single step using low concentrations of reagent. Although free cysteines are also modified by the coupling reaction, they can be protected through disulfide formation and then liberated after N-terminal coupling is complete. This allows access to doubly functionalized bioconjugates that can be difficult to access using other methods.
Topics: Aminophenols; Models, Molecular; Molecular Structure; Oxidation-Reduction; Peptides; Proline; Proteins
PubMed: 24963951
DOI: 10.1021/ja500728c -
Molecules (Basel, Switzerland) Jun 2023L-Azetidine-2-carboxylic acid (AZE) is a non-protein amino acid that shares structural similarities with its proteogenic L-proline amino acid counterpart. For this...
L-Azetidine-2-carboxylic acid (AZE) is a non-protein amino acid that shares structural similarities with its proteogenic L-proline amino acid counterpart. For this reason, AZE can be misincorporated in place of L-proline, contributing to AZE toxicity. In previous work, we have shown that AZE induces both polarization and apoptosis in BV2 microglial cells. However, it is still unknown if these detrimental effects involve endoplasmic reticulum (ER) stress and whether L-proline co-administration prevents AZE-induced damage to microglia. Here, we investigated the gene expression of ER stress markers in BV2 microglial cells treated with AZE alone (1000 µM), or co-treated with L-proline (50 µM), for 6 or 24 h. AZE reduced cell viability, nitric oxide (NO) secretion and caused a robust activation of the unfolded protein response (UPR) genes (ATF4, ATF6, ERN1, PERK, XBP1, DDIT3, GADD34). These results were confirmed by immunofluorescence in BV2 and primary microglial cultures. AZE also altered the expression of microglial M1 phenotypic markers (increased IL-6, decreased CD206 and TREM2 expression). These effects were almost completely prevented upon L-proline co-administration. Finally, triple/quadrupole mass spectrometry demonstrated a robust increase in AZE-bound proteins after AZE treatment, which was reduced by 84% upon L-proline co-supplementation. This study identified ER stress as a pathogenic mechanism for AZE-induced microglial activation and death, which is reversed by co-administration of L-proline.
Topics: Proline; Microglia; Azetidinecarboxylic Acid; Amino Acids; Endoplasmic Reticulum Stress
PubMed: 37375363
DOI: 10.3390/molecules28124808 -
MAbs 2020Preferential interactions of excipients with the antibody surface govern their effect on the stability of antibodies in solution. We probed the preferential interactions...
Preferential interactions of excipients with the antibody surface govern their effect on the stability of antibodies in solution. We probed the preferential interactions of proline, arginine.HCl (Arg.HCl), and NaCl with three therapeutically relevant IgG1 antibodies via experiment and simulation. With simulations, we examined how excipients interacted with different types of surface patches in the variable region (Fv). For example, proline interacted most strongly with aromatic surfaces, Arg.HCl was included near negative residues, and NaCl was excluded from negative residues and certain hydrophobic regions. The differences in interaction of different excipients with the same surface patch on an antibody may be responsible for variations in the antibody's aggregation, viscosity, and self-association behaviors in each excipient. Proline reduced self-association for all three antibodies and reduced aggregation for the antibody with an association-limited aggregation mechanism. The effects of Arg.HCl and NaCl on aggregation and viscosity were highly dependent on the surface charge distribution and the extent of exclusion from highly hydrophobic patches. At pH 5.5, both tended to increase the aggregation of an antibody with a strongly positive charge on the Fv, while only NaCl reduced the aggregation of the antibody with a large negative charge patch on the Fv. Arg.HCl reduced the viscosities of antibodies with either a hydrophobicity-driven mechanism or a charge-driven mechanism. Analysis of this data presents a framework for understanding how amino acid and ionic excipients interact with different protein surfaces, and how these interactions translate to the observed stability behavior.
Topics: Antibodies, Monoclonal; Arginine; Computer Simulation; Immunoglobulin G; Models, Chemical; Proline; Protein Aggregates; Sodium Chloride; Viscosity
PubMed: 32938318
DOI: 10.1080/19420862.2020.1816312 -
Organic & Biomolecular Chemistry Jan 2022Proline dehydrogenase (PRODH) catalyzes the first step of proline catabolism, the FAD-dependent oxidation of L-proline to Δ-pyrroline-5-carboxylate. PRODH plays a...
Proline dehydrogenase (PRODH) catalyzes the first step of proline catabolism, the FAD-dependent oxidation of L-proline to Δ-pyrroline-5-carboxylate. PRODH plays a central role in the metabolic rewiring of cancer cells, which has motivated the discovery of inhibitors. Here, we studied the inhibition of PRODH by 18 proline-like compounds to understand the structural and chemical features responsible for the affinity of the best-known inhibitor, -(-)-tetrahydro-2-furoic acid (1). The compounds were screened, and then six were selected for more thorough kinetic analysis: cyclobutane-1,1-dicarboxylic acid (2), cyclobutanecarboxylic acid (3), cyclopropanecarboxylic acid (4), cyclopentanecarboxylic acid (16), 2-oxobutyric acid (17), and (2)-oxetane-2-carboxylic acid (18). These compounds are competitive inhibitors with inhibition constants in the range of 1.4-6 mM, compared to 0.3 mM for 1. Crystal structures of PRODH complexed with 2, 3, 4, and 18 were determined. All four inhibitors bind in the proline substrate site, but the orientations of their rings differ from that of 1. The binding of 3 and 18 is accompanied by compression of the active site to enable nonpolar contacts with Leu513. Compound 2 is unique in that the additional carboxylate displaces a structurally conserved water molecule from the active site. Compound 18 also destabilizes the conserved water, but by an unexpected non-steric mechanism. The results are interpreted using a chemical double mutant thermodynamic cycle. This analysis revealed unanticipated synergism between ring size and hydrogen bonding to the conserved water. These structure-affinity relationships provide new information relevant to the development of new inhibitor design strategies targeting PRODH.
Topics: Dose-Response Relationship, Drug; Enzyme Inhibitors; Humans; Molecular Structure; Proline; Proline Oxidase; Structure-Activity Relationship
PubMed: 35018940
DOI: 10.1039/d1ob02328d