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Schiff Base in Enzyme Mechanisms

Schiff Base in Enzyme Mechanisms


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Can a schiff base stabilize the transition state and form an intermediate as in covalent catalysis with a substrate in the same reaction? Basically I'm wondering if a schiff base creates a ping pong reaction.


I think the answer to this one is a most definite 'yes'. Almost all aminotransferases proceed via a ping-pong mechanism where a schiff base involving pyridoxal phosphate is an obligate intermediate, and where the 'half-reation' modified enzyme form has often been isolated. An example is aspartate aminotransferase.

Let's also remind ourselves that a transition state is not an intermediate on the reaction pathway (in the TS bonds are in the process of being broken and formed), but may resemble one.


A3. Covalent or Nucleophilic Catalysis

  • Contributed by Henry Jakubowski
  • Professor (Chemistry) at College of St. Benedict/St. John's University

One way to change the activation energy of the reaction is to change the reaction mechanism in ways which introduces new steps with lower activation energy. A typical way is to add a nucleophilic catalyst which forms a covalent intermediate with the reactant. The original nucleophile can then interact with the intermediate in a nucleophilic substitution reaction. If the nucleophilic catalyst is a better nucleophile than the original nucleophile (usually water) then the reaction is catalyzed. The nucleophilic catalyst and the original nucleophile usually interact with a carbonyl C in a substitution reaction, initially forming the tetrahedral oxyanion intermediate.

Figure: NUCLEOPHILIC COVALENT CATALYSIS BY PYRIDINE.

If an amine is used as the nucleophilic catalyst, then the initial addition product (a carbinolamine) can become dehydrated, since the free pair of electrons on the N are more likely to be shared with the carbon to form a double bond than electrons from the original carbonyl O, which is more electronegative than the N). An imine or Schiff Base forms , with a pKa of about 7.

Figure: MECHANISM OF SCHIFF BASE FORMATION

This is easily protonated to form a positively charged N at the former carbonyl O center. This serves as an excellent electron sink for decarboxylation reactions of beta-keto acids and illustrates an important point. Electrons in chemical reactions can be viewed as flowing from a source (such as a carboxyl group) to a sink (such as an nucleophilic carbonyl O or a positively charged N in a Schiff base).

Figure: MECHANISM OF NUCLEOPHILIC CATALYSIS BY AMINES - SCHIFF BASE FORMATION.

Figure: ELECTRON FLOW: SOURCE TO SINK

In a subsequent section, we will discuss how protein enzymes use these same catalytic strategies. An intriguing question arises: how much of the structure of a large protein is really needed for catalysis? Much work has been directed to the development of small molecule catalysis mimetics of large protein enzymes. Just how small can you go in reducing the size of a protein and still get catalysis. One important feature of enzyme catalysis is that they catalyze reactions in which only one enantiomer is produced. That is, the synthesis is assymertric. This is typically a consequence of the asymmetric enzyme (itself chiral) binding only one enantiomer as a reactant and/or the imposition of steric restrictions on the possible reactions of the bound substrate. Recently, it has been show that L-Pro alone can act as such an assymetric catalyst in an aldol condensation reaction.

Figure: L-PRO CATALYSIS OF AN ALDOL CONDENSATION: POSSIBLE MECHANISM


PYRIDOXAL PHOSPHATE ENZYMES

Pyridoxal phosphate (PLP) is a derivative of the vitamin B6 or pyridoxal. Deficiencies cause convulsions, chronic anemia, and neuropathy. It assists in many reactions (catalyzed by PLP-dependent enzymes). The PLP is bound covalently to lysine residues in a Schiff base linkage (aldimine). In this form, it reacts with many free amino acids (as substrates) to replace the Schiff base to Lys of the enzyme with a Schiff base to the amino acid substrate. First a review of Schiff Base formation.

PLP: Structure and Covalent Attachment to Enzyme

For reactions 6-8, assume that the amino acid substrate is in a Schiff base with PLP.

William Jencks, in his classic text, Catalysis in Chemistry, wrote:

"It has been said that God created an organism especially adapted to help the biologist find an answer to every question about the physiology of living systems if this is so, it must be concluded that pyridoxal phosphate was created to provide satisfaction and enlightenment to those enzymologists and chemists who enjoy pushing electrons, for no other coenzyme is involved in such a wide variety of reactions, in both enzyme and model systems, which can be reasonably interpreted in terms of the chemical properties of the coenzyme. Most of these reactions are made possible by a common structural feature. That is, electron withdrawal toward the cationic nitrogen atom of the imine and into the electron sink of the pyridoxal ring from the alpha carbon atom of the attached amino acid activates all three of the substitutents of this carbon for reactions which require electron withdrawal from this atom."

Molecular Modeling: PLP: Tyrosine Aminotransferase Jmol (from PDB)

6. RX TYPE - &alpha -DECARBOXYLATION OF AMINO ACIDS.

7. RX TYPE - BETA-ELIMINATION FROM SERINE. Example: Serine dehydratase. (hint: remove H on &alpha-C first), then OH)

8. RX TYPE - RACEMIZATION OF AMINO ACIDS. (hint: remove H on &alpha-C first)

PLP enzymes also catalyze transamination reactions, an example of which is shown below:

Amino Acid 1 + &alpha-keto acid 1 <==> &alpha-keto acid 2 + Amino Acid 2 For example:

First Asp, bound to PLP through a Schiff base link, loses the &alpha-H , forms a ketimine through a tautomerization reactions, which ultimately hydrolyzes to form the released oxalacetate and pyridoxamine. The pryidoxamine reacts with &alpha-ketoglutarate in the reverse of the first three reactions to from Glu.

9. RX TYPE - ACETYLATION: The "acetic anhydride" of biological acetylation reactions is acetyl-CoA, a derivative of the vitamin pantathenic acid, which contains a free thiol. It is acetylated at the thiol in many metabolic reactions to produce acetylCoA containing a thioester bond, a biological acetylating reagent. This molecule can be cleaved in an exergonic fashion due in part to the weak bond between the acetyl C and the Sk leading to transfer of the acetyl group. We have previously discussed the importance of histone Lys acetylation by histone acetylases in the control of gene expression.


Structure and mechanism of a sub-family of enzymes related to N-acetylneuraminate lyase.

We describe here a sub-family of enzymes related both structurally and functionally to N-acetylneuraminate lyase. Two members of this family (N-acetylneuraminate lyase and dihydrodipicolinate synthase) have known three-dimensional structures and we now proceed to show their structural and functional relationship to two further proteins, trans-o-hydroxybenzylidenepyruvate hydratase-aldolase and D-4-deoxy-5-oxoglucarate dehydratase. These enzymes are all thought to involve intermediate Schiff-base formation with their respective substrates. In order to understand the nature of this intermediate, we have determined the three-dimensional structure of N-acetylneuraminate lyase in complex with hydroxypyruvate (a product analogue) and in complex with one of its products (pyruvate). From these structures we deduce the presence of a closely similar Schiff-base forming motif in all members of the N-acetylneuraminate lyase sub-family. A fifth protein, MosA, is also confirmed to be a member of the sub-family although the involvement of an intermediate Schiff-base in its proposed reaction is unclear.


The mechanism of the glycosylase reaction with hOGG1 base-excision repair enzyme: concerted effect of Lys249 and Asp268 during excision of 8-oxoguanine

The excision of 8-oxoguanine (oxoG) by the human 8-oxoguanine DNA glycosylase 1 (hOGG1) base-excision repair enzyme was studied by using the QM/MM (M06-2X/6-31G(d,p):OPLS2005) calculation method and nuclear magnetic resonance (NMR) spectroscopy. The calculated glycosylase reaction included excision of the oxoG base, formation of Lys249-ribose enzyme-substrate covalent adduct and formation of a Schiff base. The formation of a Schiff base with ΔG# = 17.7 kcal/mol was the rate-limiting step of the reaction. The excision of the oxoG base with ΔG# = 16.1 kcal/mol proceeded via substitution of the C1΄-N9 N-glycosidic bond with an H-N9 bond where the negative charge on the oxoG base and the positive charge on the ribose were compensated in a concerted manner by NH3+(Lys249) and CO2-(Asp268), respectively. The effect of Asp268 on the oxoG excision was demonstrated with 1H NMR for WT hOGG1 and the hOGG1(D268N) mutant: the excision of oxoG was notably suppressed when Asp268 was mutated to Asn. The loss of the base-excision function was rationalized with QM/MM calculations and Asp268 was confirmed as the electrostatic stabilizer of ribose oxocarbenium through the initial base-excision step of DNA repair. The NMR experiments and QM/MM calculations consistently illustrated the base-excision reaction operated by hOGG1.

© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Figures

The hOGG1 BER enzyme. Sketch…

The hOGG1 BER enzyme. Sketch of the QM/MM structural model including hOGG1 (cyan)…

The local geometries of the…

The local geometries of the catalytic core calculated for stationary states of the…

The chemical diagram ( A…

The chemical diagram ( A ) and free-energy profile ( B ) of…

The catalytic function of WT…

The catalytic function of WT hOGG1 and hOGG1(D268N). Concept of the assay for…

The QM/MM-calculated two-dimensional potential energy…

The QM/MM-calculated two-dimensional potential energy surfaces for excision of the oxoG base with…

Summary for the base-excision reaction…

Summary for the base-excision reaction with hOGG1: compatibility of the charged residues within…


Abstract

Phosphonoacetaldehyde hydrolase (phosphonatase) catalyzes the hydrolysis of phosphonoacetaldehyde to acetaldehyde and inorganic phosphate. In this study, the genes encoding phosphonatase in Bacillus cereus and in Salmonella typhimurium were cloned for high-level expression in Escherichia coli. The kinetic properties of the purified, recombinant phosphonatases were determined. The Schiff base mechanism known to operate in the B. cereus enzyme was verified for the S. typhimurium enzyme by phosphonoacetaldehyde−sodium borohydride-induced inactivation and by site-directed mutagenesis of the catalytic lysine 53. The protein sequence inferred from the B. cereus phosphonatase gene was determined, and this sequence was used along with that from the S. typhimurium phosphonatase gene sequence to search the primary sequence databases for possible structural homologues. We found that phosphonatase belongs to a novel family of hydrolases which appear to use a highly conserved active site aspartate residue in covalent catalysis. On the basis of this finding and the known stereochemical course of phosphonatase-catalyzed hydrolysis at phosphorus (retention), we propose a mechanism which involves Schiff base formation with lysine 53 followed by phosphoryl transfer to aspartate (at position 11 in the S. typhimurium enzyme and position 12 in the B. cereus phosphonatase) and last hydrolysis at the imine C(1) and acyl phosphate phosphorus.

This work was supported by NIH Grants GM-36360 (D.D.-M.) and GM-35392 (B.L.W.) and Department of Energy Grant DE-FG03-96ER62269 (P.C.B.).

Current address: Department of Microbiology, University of Illinois, Urbana, IL 61801-3704.

National Institutes of Health.

Corresponding author. E-mail: [email protected] Telephone: (505) 277-3383. Fax: (505) 277-6202.


Schiff Base in Enzyme Mechanisms - Biology

Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.85 Å
  • R-Value Free: 0.187 
  • R-Value Work: 0.149 
  • R-Value Observed: 0.149 

wwPDB Validation   3D Report Full Report

Mechanism of the Schiff base forming fructose-1,6-bisphosphate aldolase: structural analysis of reaction intermediates.

(2005) Biochemistry 44: 4222-4229

  • PubMed: 15766250  Search on PubMed
  • DOI: 10.1021/bi048192o
  • Primary Citation of Related Structures:  
    2YCE, 1W8S
  • PubMed Abstract: 

The glycolytic enzyme fructose-1,6-bisphosphate aldolase (FBPA) catalyzes the reversible cleavage of fructose 1,6-bisphosphate to glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. Catalysis of Schiff base forming class I FBPA relies on a number of intermediates covalently bound to the catalytic lysine .

The glycolytic enzyme fructose-1,6-bisphosphate aldolase (FBPA) catalyzes the reversible cleavage of fructose 1,6-bisphosphate to glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. Catalysis of Schiff base forming class I FBPA relies on a number of intermediates covalently bound to the catalytic lysine. Using active site mutants of FBPA I from Thermoproteus tenax, we have solved the crystal structures of the enzyme covalently bound to the carbinolamine of the substrate fructose 1,6-bisphosphate and noncovalently bound to the cyclic form of the substrate. The structures, determined at a resolution of 1.9 A and refined to crystallographic R factors of 0.148 and 0.149, respectively, represent the first view of any FBPA I in these two stages of the reaction pathway and allow detailed analysis of the roles of active site residues in catalysis. The active site geometry of the Tyr146Phe FBPA variant with the carbinolamine intermediate supports the notion that in the archaeal FBPA I Tyr146 is the proton donor catalyzing the conversion between the carbinolamine and Schiff base. Our structural analysis furthermore indicates that Glu187 is the proton donor in the eukaryotic FBPA I, whereas an aspartic acid, conserved in all FBPA I enzymes, is in a perfect position to be the general base facilitating carbon-carbon cleavage. The crystal structure of the Trp144Glu, Tyr146Phe double-mutant substrate complex represents the first example where the cyclic form of beta-fructose 1,6-bisphosphate is noncovalently bound to FBPA I. The structure thus allows for the first time the catalytic mechanism of ring opening to be unraveled.


INTRODUCTION

The repair of damaged DNA is necessary to preserve genome integrity, and the damaged DNA bases are eliminated with the base-excision repair (BER) enzymes ( 1– 5). The excision of the damaged base and scission of the DNA strand that involves abasic site are operated by bi-functional BER enzymes within the glycosylase and subsequent β-lyase reaction, respectively ( 6). The excision of the corrupted DNA base is the first irreversible step of the repair pathway that is initiated upon formation of the enzyme–DNA complex involving structural rearrangement of the corrupted DNA strand (Figure 1A) ( 7– 9).

The hOGG1 BER enzyme. Sketch of the QM/MM structural model including hOGG1 (cyan) and oxoG-containing DNA (red) that was derived from the 2NOZ ( 33) crystal structure (A). The chemical structure of guanine (G), oxoG, Lys249 and Asp268 in WT hOGG1 and Asn268 in the hOGG1(D268N) mutant (B). Detail of the catalytic core before oxoG excision in 2NOZ (yellow) and in the QM/MM-optimized reactant (atom colored) (C). Detail of the catalytic core including Lys249-ribose covalent adduct with opened ring of sugar in the 1HU0 ( 34) crystal (yellow) and in the QM/MM-calculated reaction product (stationary state P+1) describing the Schiff base (atom colored) (D).

The hOGG1 BER enzyme. Sketch of the QM/MM structural model including hOGG1 (cyan) and oxoG-containing DNA (red) that was derived from the 2NOZ ( 33) crystal structure (A). The chemical structure of guanine (G), oxoG, Lys249 and Asp268 in WT hOGG1 and Asn268 in the hOGG1(D268N) mutant (B). Detail of the catalytic core before oxoG excision in 2NOZ (yellow) and in the QM/MM-optimized reactant (atom colored) (C). Detail of the catalytic core including Lys249-ribose covalent adduct with opened ring of sugar in the 1HU0 ( 34) crystal (yellow) and in the QM/MM-calculated reaction product (stationary state P+1) describing the Schiff base (atom colored) (D).

The primary factor leading to accumulation of DNA mutations is oxidative stress. oxoG, which arises from oxidatively damaged guanine (G) belongs to the most abundant and most dangerous DNA lesions (Figure 1B) ( 10– 12). oxoG induces serious defects to organisms, and its continuous removal is crucial for the elimination of unwanted consequences of basal respiratory processes that normally occur in cells ( 13). By contrast, the controlled shutoff of oxoG repair in cancer cells is assumed to be remedial in advanced anticancer therapies ( 14).

The glycosylase reaction operated by BER enzymes requires activation to surmount the energy barrier for cleavage of the N-glycosidic bond. The supply of up to 19 kcal/mol accelerates the bond cleavage 10 14 -fold ( 4). All of the BER enzymes are typically lesion-specific which is particularly true for the hOGG1, which operates against oxoG with astonishing specificity ( 15– 28). Misbehavior of hOGG1 most likely cannot occur, as a catalytic checkpoint prevents even scission of G forcibly inserted into the catalytic site ( 29). The insertion of oxoG into the catalytic site occurs much faster than the subsequent rearrangement of catalytic core ( 30). An inhibitor of hOGG1 that can mimic true substrates thus could become a therapeutic agent against certain types of cancer ( 31).

The structure of the hOGG1 catalytic core was disclosed by Verdine's group. First, they revealed the mechanistic basis for recognition and excision of oxoG using the inactive hOGG1(K249Q) mutant, where the Lys249 was substituted with Gln ( 8). Later, they demonstrated that Asp268 is another principal catalytic residue using the hOGG1(D268N) mutant where the Asp268 was substituted with Asn (Figure 1B) ( 32). These results led us to the focus on Lys249 and Asp268 in our work. Gly42, depicted also in the Figure 1B, is presumably responsible for recognition and proper deposition of oxoG within the catalytic core by the force of a hydrogen bond with H7(oxoG) ( 29).

The structure of the hOGG1 catalytic core, just before oxoG excision, was captured in the 2NOZ crystal (Figure 1C). Several glycosylase reaction mechanisms were assumed for different protonation states of Asp268 and Lys249 because the actual forms are currently unknown. CO2 − (Asp268) was assumed to ensure electrostatic stabilization of the oxocarbenium cation on ribose during rupture of the N-glycosidic bond, as the mutation of Asp268 to Asn notably suppressed function of the hOGG1(D268N) mutant ( 32, 35). As for Lys249, similar closeness of the Nε nitrogen to N3, N9 and C1΄ atoms of oxoG (Figure 1B), observed in the crystals, indicated a variety of base-excision reactions. NH3 + (Lys249) could stabilize the negative charge on the leaving oxoG base, whereas NH2(Lys249) could stabilize the oxocarbenium cation on sugar. The respective base-excision reactions had actually been assumed ( 6, 23, 32, 34, 36, 37). The mutation of Lys249 to Cys or Gln caused a loss of catalytic function of the bi-functional hOGG1 ( 8, 34, 37). Lys249 forms a Schiff base with the ribose of abasic site after oxoG excision ( 8). The Lys249-ribose covalent adduct, generated from the Schiff base upon the addition of a reducing agent, was captured in the 1HU0 crystal ( 34). Importantly, the geometries of the reactant and the product captured in the crystals can be used for reliable theoretical modeling of the glycosylase reaction (Figure 1C and D).

Compensation for the developing charges within a molecule of substrate by principal catalytic residues is a typical enzyme strategy ( 38). This general strategy was considered in theoretical calculations of the base-excision reactions operated by hOGG1 in a number of studies. The effect of NH2(Lys249) on the oxocarbenium cation of ribose was compared with the effects of other nucleophiles ( 39, 40). The cascade migration of the proton from NH3 + (Lys249) to O8(oxoG) and then to O4΄ of ribose activated the opening of the ribose ring and excision of the oxoG base ( 41). The synchronous attack of NH2(Lys249) to C1΄(ribose) and CO2H(Asp268) to O4΄(ribose) resulted in an opened ring of the oxoG ribose and subsequent excision of the oxoG base (the Ribose-protonated mechanism) ( 42). A similar mechanism was proposed for the opening of the sugar ring of a substrate nucleoside with endonuclease III-DNA ( 43). The π–cation interaction between the aromatic ring of the oxoG base and NH3 + (Lys249) initiated proton transfer from NH3 + (Lys249) to N3(oxoG), which activated the base-excision reaction ( 44, 45). Lastly, the abstraction of the proton from NH3 + (Lys249) with the N9(oxoG) atom triggered substitution of the N-glycosidic bond of oxoG with the N9-H bond in a concerted synchronous manner (the σ-bond substitution mechanism) ( 46).

The previous studies clearly demonstrate the principal effect of Lys249 and Asp268 on the catalytic function of hOGG1. As mentioned, protonation states of the two residues are presently unknown, and the function of these residues during the glycosylase reaction is therefore obscure. Nevertheless, typical pKa values for the Lys and Asp side-chains implicate likely NH3 + (Lys249) and CO2 − (Asp268) forms ( 47). Under this assumption, Lys249 presumably compensates for the negative charge on the leaving oxoG base and Asp268 compensates for the incipient charge on the ribose oxocarbenium. The σ-bond substitution reaction that complies with these assumptions was proposed previously, assuming only the effect of Lys249 ( 46). In the current work, the base-excision reaction with hOGG1 will be illustrated within a complete catalytic core by employing the QM/MM calculation method. The function of Asp268 will be particularly addressed. The Asp residue is known to be well-conserved with within the OGG family of BER enzymes however, the function is still not clear ( 4). The nuclear magnetic resonance (NMR) measurements employing WT hOGG1 and the hOGG1(D268N) mutant in this work determined which of the functions of the bi-functional hOGG1 is actually affected by the D268N mutation. The NMR experiments and QM/MM calculations provided a coherent picture of the base-excision reaction within the glycosylase reaction operated by the hOGG1 BER enzyme.


First observation of metamorphosis of an enzyme that catalyzes two chemical reactions

Figure 2. Total three-dimensional structure of FBPA/P. Eight identical units gather to form a barrel-like structure. Each unit is represented using an iridescent color (blue, light blue, green, yellowish green, orange, and red). Bound DHAP bases and magnesium ions are depicted as purple and pink spheres, respectively.

Professor Takayoshi Wakagi and Associate Professor Shinya Fushinobu of the Graduate School of Agricultural and Life Sciences, the University of Tokyo and colleagues were the first to clarify how an enzyme of hyperthermophilic archaea origin that is thought to be positioned close to the origin of life can catalyze two reactions while metamorphosing itself.

Archaea possess many unusual enzymes. Among them, fructose-1,6-bisphosphate (FBP) aldolase/phosphatase is especially peculiar. In apparent violation of biochemical canons, this enzyme catalyzes two fundamentally different chemical reactions. Additionally, this enzyme is responsible for the biosynthesis of glucose, a process of critical importance in the early stage of the evolution of life.

The research team utilized the Photon Factory at KEK to observe the enzyme’s structural metamorphosis to bring about the catalysis of the two different reactions. Such a multifunctional enzyme shatters long-held beliefs in biochemistry and raises the intriguing possibility that more such enzymes might be discovered in other organisms.

Hyperthermophiles live in ultra-hot water and are positioned near the root of the evolutionary tree of life. As such, they are thought to be close to the common ancestor of life on Earth. Many hyperthermophiles are categorized as archaea, which belong to different branches from those of common organisms, such as bacteria and eukaryotes. Some hyperthermophilic archaea have the ability to build their own bodies by synthesizing complex compounds from simple inorganic substances, for example in the biosynthesis of saccharides from carbon dioxide. Reaction pathways in which organisms synthesize glucose are thought to be important in the evolution of primitive organisms.

Figure 3. Reactions that are catalyzed by FBPA/P (left) and schematic views of regions where reactions occur (right). Three loops undergo significant conformational changes in changing between the FBP aldolase form (top right) and the FBP phosphatase form (bottom right).

For most common organisms two different enzymes, fructose-1,6-bisphosphate (FBP) aldolase and FBP phosphatase, are sequentially responsible for chemical reactions that lead to glucose production. By contrast, hyperthermophilic archaea make use of a single protein, termed FBP aldolase/phosphatase (FBPA/P), to catalyze these two reactions. This enzyme could be said to be bifunctional.

The research team studied FBPA/P found in the hyperthermophilic archaeon, Sulfolobus, isolated from hot water at Beppu Onsen in Oita Prefecture, Japan. The three-dimensional structure of FBPA/P looks similar to a barrel of eight identical molecular units. Reactions are catalyzed in the regions between these units.

The two reactions that FBPA/P catalyzes are: 1) FBP aldolase reaction, in which dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (GA3P) produce FBP and 2) FBP phosphatase reaction, in which FBP is cleaved into fructose-6-phosphate (F6P) and inorganic phosphate (Pi).

In 2004, the research team elucidated the three-dimensional structure of the enzyme-substrate complex while FBPA/P is catalyzing the FBP phosphatase reaction. In the present study, the same group succeeded in unraveling the three-dimensional form of the enzyme-substrate complex while FBPA/P is catalyzing the FBP aldolase reaction by using X-ray crystallography in their experiments performed at the AR-NW12A beamline of the KEK’s Photon Factory. Comparison of these two forms revealed that the active site (the region of an enzyme in which a chemical reaction is catalyzed) of FBPA/P metamorphoses into two completely different forms owing to the extensive movements of three loops (lid loop, Schiff-base loop, and C-terminal loop).

In the FBP aldolase form, a lysine residue (K232) binds to the first substrate, DHAP. When the second substrate, GA3P, enters the active center, a neighboring tyrosine residue (Y229) is thought to mediate reaction of GA3P and DHAP into FBP (FBP aldolase reaction). Once FBP is produced, the lysine residue becomes free and the three loops can move. The Schiff-base loop is flipped over and both the lid and C-terminal loops close, metamorphosing the enzyme into its FBP phosphatase form. Next, an aspartate residue (D233) enters the active center, and binds magnesium ions, thereby triggering the dissociation of FBP into F6P and Pi (FBP phosphatase reaction).

This finding revealed that such a ‘metamorphosis’ is the key to the catalysis of two different reactions for a given enzymatic reaction sequence. This mechanism turns on its head the established biochemical dogma that describes one enzyme as catalyzing only one reaction.

Common organisms of more recent ancestry do not possess FBPA/P, but rather make use of two different enzymes separately to catalyze the two reactions independently. It is possible that primitive organisms might perform biosynthesis in a simpler manner than more contemporary organisms do by utilizing bifunctional enzymes such as FBPA/P.

The present study is the first to reveal the mechanism by which one enzyme may catalyze two different chemical reactions. Moreover, the current finding hints at the possibility that more multifunctional enzymes similar to FBPA/P may exist. In addition, it can be expected that more enzymes capable of converting simple starting materials into synthetically useful intermediates will be discovered.


A Novel Thiazolyl Schiff Base: Antibacterial and Antifungal Effects and In Vitro Oxidative Stress Modulation on Human Endothelial Cells

Schiff bases (SBs) are chemical compounds displaying a significant pharmacological potential. They are able to modulate the activity of many enzymes involved in metabolism and are found among antibacterial, antifungal, anti-inflammatory, antioxidant, and antiproliferative drugs. A new thiazolyl-triazole SB was obtained and characterized by elemental and spectral analysis. The antibacterial and antifungal ability of the SB was evaluated against Gram-positive and Gram-negative bacteria and against three Candida strains. SB showed good antibacterial activity against L. monocytogenes and P. aeruginosa it was two times more active than ciprofloxacin. Anti-Candida activity was twofold higher compared with that of fluconazole. The effect of the SB on cell viability was evaluated by colorimetric measurement on cell cultures exposed to various SB concentrations. The ability of the SB to modulate oxidative stress was assessed by measuring MDA, TNF-α, SOD1, COX2, and NOS2 levels in vitro, using human endothelial cell cultures exposed to a glucose-enriched medium. SB did not change the morphology of the cells. Experimental findings indicate that the newly synthetized Schiff base has antibacterial activity, especially on the Gram-negative P. aeruginosa, and antifungal activity. SB also showed antioxidant and anti-inflammatory activities.

1. Introduction

Aerobic organisms have antioxidant defense systems against reactive oxygen species- (ROS-) induced damage produced in various stress conditions. ROS are also involved in the innate immune system and have an important role in the inflammatory response they attract cells, by chemotaxis, to the inflammation site. Nitric oxide (NO) is another important intracellular and intercellular signaling molecule involved in the regulation of multiple physiological and pathophysiological mechanisms. It acts as a biological modulator. NO is able to regulate vascular tone and can function as a host defense effector. Also, it can act as a cytotoxic agent in inflammatory disorders. NO synthase (NOS) enzyme family catalyzes NO production. Inhibition of inducible NOS (iNOS) might be beneficial in the course of treatment of certain inflammatory diseases [1]. The reactions between NO and ROS, such as superoxide radicals (O2 ⋅− ), lead to the production of a potent prooxidant radical (peroxynitrite), thus inducing endothelial and mitochondrial dysfunction. The major cellular defense against peroxide and peroxynitrite radicals are the superoxide dismutases (SODs) that catalyzes the transformation of peroxide radicals into hydrogen peroxide (H2O2), which is further transformed by catalase into water and molecular oxygen. Also, SODs play an important role in preventing peroxynitrite formation [2]. All isoforms have in their catalytic site a transition metal, such as copper and manganese [3].

Recent studies showed that exogenous NO, produced by bacterial NOS, protects Gram-positive and Gram-negative bacteria (Pseudomonas aeruginosa, Staphylococcus aureus, etc.) against oxidative stress and increases bacterial resistance to a broad spectrum of antibiotics [4]. Fungal resistance to antimycotic treatment is one of the consequences of the emergence of resistant strains, but more and more, in the last years, fungal resistance is due to the capacity of fungal strains to form biofilms, which are considered critical in invasive fungal infections, associated with high mortality. Certain studies showed that only a few antimycotics are effective against fungal biofilms. All of them have the capacity to induce ROS formation in fungal biofilm cells [5]. In this context, finding bioactive substances capable to reduce NO synthesis in bacteria or able to induce ROS synthesis in fungal biofilms could represent new directions in the development of new antimicrobial drugs. Thiazoles, triazoles, and their derivatives are found among antibacterial and anti-inflammatory drugs [6–9].

Schiff bases (SBs) are chemical structures that have a significant pharmacological potential. SBs contain an azomethine group obtained through the condensation of primary amines with carbonyl compounds [10]. The pharmacophore potential of this group is due to their ability to form complex compounds with bivalent and trivalent metals located in the active center of numerous enzymes involved in metabolic reactions. The relationship between a chemical structure and biological activity (SAR) underlines the importance of the azomethine group for the synthesis of new compounds with antibacterial, antifungal, and even antitumor activities [11–13].

Multiple studies showed the ability of SBs to act as antiproliferative and antitumoral agents [14–16]. The azomethine pharmacophore is used in developing new bioactive molecules [17]. The discovery of selective cytotoxic drugs influenced oncological therapy. However, completely satisfactory answers for metastasis onset have not yet been found. Due to the increased prevalence of neoplasia and to the existence of various cellular tumor lines resistant to cytotoxic therapy, the research of new active agents is justified [18, 19].

The current study is aimed at testing a newly synthetized heterocyclic SB in terms of antimicrobial activity against Gram-positive and Gram-negative bacteria and antifungal effects against Candida strains [20, 21], as well as to evaluate the biocompatibility of the SB in vitro on human endothelial cells and the ability of this SB to modulate oxidative stress, by assessing enzymes involved in cellular antioxidant defense.

2. Materials and Methods

2.1. Synthesis of the Schiff Base

All reagents and solvents used were purchased from Sigma-Aldrich and were used without further purification. The starting compound was previously reported and was synthesized by us according to methodologies described in the literature [21].

The synthesis of Schiff base (SB) 4-(3-bromobenzylideneamino)-5-(4-methyl-2-phenylthiazol-5-yl)-4H-1,2,4-triazole-3-thiol was made using a general procedure (Scheme 1) [21]. 2 mmol (0.578 g) of 4-amino-5-(4-methyl-2-phenylthiazol-5-yl)-4H-1,2,4-triazole-3-thiol was suspended in 10 mL of absolute ethanol. The resulting suspension was added with an alcoholic solution of 2 mmol of 3-bromobenzaldehyde in 5 mL of absolute ethanol and 2-3 drops of concentrated H2SO4, as a catalyst. The reaction mixture was refluxed for 6 h. The obtained precipitate was filtered hot and washed with absolute ethanol, and then, it was dried and recrystallized from dimethyl sulfoxide (DMSO).

2.2. In Vitro Antibacterial and Antifungal Screening
2.2.1. Preparation of Sample Solution

SB was dissolved in DMSO, at a final concentration of 100 μg/mL. Sample solution was stored at 4°C [22, 23].

2.2.2. Inhibition Zone Diameter Measurements

Antimicrobial activity was tested in vitro using the agar disk diffusion method through the measurement of the inhibition zone diameters. Agar plates were inoculated with a standardized inoculum of the test microorganisms: two Gram-negative bacterial strains—Salmonella enteritidis ATCC 14028 and Escherichia coli ATCC 25922, two Gram-positive bacterial strains—Listeria monocytogenes ATCC 19115 and Staphylococcus aureus ATCC 49444, and a fungal strain—Candida albicans ATCC 10231. Petri plates with Mueller Hinton Agar (20.0 mL) were used for all bacterial tests. Mueller-Hinton medium supplemented with 2% glucose (providing adequate growth of yeasts) and 0.5 g/L methylene blue (providing a better definition of the inhibition zone diameter) was used for antifungal testing. Each paper disk was impregnated with 10 μL of solution (100 μg compound/disk). The filter paper disks were placed on Petri dishes previously seeded “in layer” with the tested bacterial strain inoculums. Then, Petri dishes were maintained at room temperature to ensure the equal diffusion of the compound in the medium, and afterwards, the dishes were incubated at 37°C for 24 hours. Inhibition zones were measured after 24 hours of incubation. Assessment of the antimicrobial effect was realized by measuring the diameter of the growth inhibition zone. Ciprofloxacin (10 μg/well) and fluconazole (25 μg/well) were used as standard antibacterial and antifungal drugs. DMSO was used for comparison, as a negative control, for all experiments, and it did not inhibit the growth of microorganisms (

). The clear halos with a diameter larger than 10 mm were considered positive results [22, 23]. Tests were performed in triplicate, and values are presented as the

2.2.3. Determination of Minimum Inhibitory Concentrations (MICs), Minimum Bactericidal Concentrations (MBCs), and Minimum Fungicidal Concentrations (MFCs)

Minimum inhibitory concentrations (MICs), minimum bactericidal concentrations (MBCs), and minimum fungicidal concentrations (MFCs) were determined by an agar dilution method. Strains of microorganisms used were as follows: Salmonella enteritidis ATCC 14028, Escherichia coli ATCC 25922, Listeria monocytogenes ATCC 19115, Staphylococcus aureus ATCC 49444, Candida albicans ATCC 10231, Candida albicans (ATCC 18804), and Candida krusei (ATCC 6258) [22–26]. For the experiment, 100 μL nutrient broths were placed in a 96-well plate, and sample solution at high concentration (100 μg/mL) was added into the first rows of the microplates. 10 μL of culture suspensions was inoculated into all the wells. The plates were incubated at 37°C for 16-24 hours (48 hours for fungi). The reference drugs, ciprofloxacin and fluconazole, were used in the same concentrations.

2.3. Determination of Antioxidant Activity by DPPH (2,2-Diphenyl-1-picrylhydrazyl) Bleaching Assay

The DPPH antioxidant activity assay was done as previously described, with minor modification. SB was dissolved in DMSO (1 mg/mL). DPPH∙ radical was dissolved in methanol (0.25 mM). Equal volumes (1.0 mL) of methanolic DPPH solution and sample solution (or standard) in methanol at different concentrations have been used. The mixtures were incubated for 30 min at 40°C in a thermostatic bath absorbance was measured at 517 nm. The percent DPPH scavenging ability was calculated as follows:

, where is the absorbance of DPPH radical and methanol (containing all reagents, except the sample) and is the absorbance of the mixture of DPPH radical and sample. A curve of % DPPH scavenging ability versus concentration was plotted, and IC50 values were calculated. The IC50 value is the sample concentration required to scavenge 50% of DPPH free radicals. The lesser the IC50 value, the stronger the antioxidant capacity. Thus, if

, the sample shows a high antioxidant capacity if

, the sample has a moderate antioxidant capacity if

, the sample has poor or no activity. BHT (butylated hydroxytoluene) and trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) were used as positive controls [20, 27–30].

2.4. Assessment of the Ability of the SB to Modulate Inflammatory Response and Oxidative Stress on Cell Cultures
2.4.1. Cell Source

Human umbilical vein endothelial cells (HUVECs, Promocell, Hamburg, Germany) were used. The cells were grown in RPMI medium supplemented with 5% fetal calf serum, 50 μg/mL gentamycin, and 5 ng/mL amphotericin (Biochrom Ag, Berlin, Germany). Cell cultures in the 13 rd to 15 th passages were used. SB was diluted in DMSO (Biochrom Ag, Berlin, Germany) to obtain a stock solution of 1 mg/mL. The stock solution was used to make further dilutions in complete cell growth medium, immediately prior to the experiments. The final DMSO concentration was lower than 0.05%, a nontoxic concentration for the cells [31].

2.4.2. Cell Viability Testing

Cells cultured at a density of 10 4 /well on ELISA 96-well plaques (TPP, Switzerland) were settled for 24 hours, then exposed to different concentrations of the substance ranging from 0.001 to 200 μg/mL. Viability was measured by the colorimetric measurement of a colored compound—formazan, generated by viable cells using the CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay (Promega Corporation, Madison, USA). Readings were done at 540 nm, using an ELISA plate reader (Tecan, Mannedorf, Austria). Results were presented as OD540. All experiments were performed in triplicate. Untreated cultures were used as controls [32].

2.4.3. Experimental Design

Four groups were made: (1) control cells treated only with medium, (2) cells exposed to a high-glucose (4.5 g/L) medium, (3) cells treated with SB 0.001 μg/mL, and (4) cells concomitantly exposed to high glucose and SB (0.001 μg/mL). All groups were treated for 24 hours. Afterwards, cells were used for the assessment of cytoskeleton modifications—phalloidin staining (fluorescence microscopy), oxidative stress (Western blot measurement of SOD1, COX2, and inducible NOS2 and spectrophotometric measurement of MDA), and inflammation (ELISA measurement of TNF-α).

2.4.4. Cell Lysis

The cell lysates used in the following experiments were prepared as previously described [33]. Protein concentrations were determined by the Bradford method, according to the manufacturer’s specifications (Bio-Rad, Hercules, California, USA) and using bovine serum albumin as standard. For all assays, the lysates were corrected by total protein concentration.

2.4.5. Oxidative Stress and Inflammation Assessment

Quantification of malondialdehyde (MDA) a marker for the peroxidation of membrane lipids was performed by spectrophotometry, as previously described [34]. All reagents were purchased from Sigma-Aldrich. Data were expressed as nM/mg protein [35]. Following viability testing and following the assessment of the MDA level, cells used in further experiments were treated with a concentration of 0.001 μg/mL.

TNF-α ELISA Immunoassay kit from R&D Systems, Inc. (Minneapolis, USA) was used. Cell supernatants were treated according to the manufacturer’s instructions readings were done at 450 nm with correction wavelength set at 540 nm, using an ELISA plate reader (Tecan) [33].

Lysates (20 μg protein/lane) were separated by electrophoresis on SDS PAGE gels and transferred to polyvinylidene difluoride membranes, using a Bio-Rad Miniprotean system (Bio-Rad). Blots were blocked and then incubated with antibodies against superoxide dismutase 1 (SOD1), cyclooxygenase 2 (COX2), and inducible nitric oxide synthase 2 (NOS2), then further washed and incubated with corresponding secondary peroxidase-linked antibodies. All antibodies were acquired from Santa Cruz Biotechnology. Proteins were detected using Supersignal West Femto Chemiluminescent substrate (Thermo Fisher Scientific, Rockford IL, USA) and a Gel Doc Imaging system equipped with a XRS camera and Quantity One analysis software (Bio-Rad). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Trevigen Biotechnology, Gaithersburg, MD (Maryland), USA) was used as a protein loading control.

Phalloidin-FITC 50 μg/mL (Sigma-Aldrich, St. Louis, MO, USA) a marker for actin myofilaments (green) was used, according to the manufacturer’s instructions. Cells were seeded in chamber slides at a density of

/chamber, allowed to settle for 24 hours, and then exposed to high glucose and SB as described above. Treated cells were then stained with phalloidin-FITC. Images of cells were documented at a magnification of 20x, using an inverted microscope Olympus BX40 equipped with an Olympus CKX-RFA fluorescent lamp and an E330 camera (Olympus, Hamburg, Germany).

2.5. Statistical Analysis

The statistical significance of the differences between the control group and the treated groups was assessed with the nonparametric Kruskal-Wallis test for multiple groups, followed by a post hoc analysis using the Conover test. Correlation coefficients between parameters have been calculated using Spearman’s correlation coefficient for ranks (rho). Statistical tests were performed using MedCalc version 18.11.3 and GraphPad Prism Software version 8.0.2. The results were considered statistical significant at

3. Results

3.1. Chemical Characterization of the SB

The SB structure was confirmed by elemental analysis and on the basis of its mass spectrum (MS), infrared spectrum (IR), and nuclear magnetic resonance ( 1 H NMR and 13 C NMR) spectra [21].

4-(3-Bromobenzylideneamino)-5-(4-methyl-2-phenylthiazol-5-yl)-4H-1,2,4-triazole-3-thiol. Yield 80.3% (0.366 g) m.p. 268-270°C light yellow powder Anal. Calcd for C19H14BrN5S2 (456.38): C, 49.89 H, 3.06 N, 15.33 S, 14.02 Found: C, 50.1 H, 3.07 N, 15.33 S, 14.07 IR (ATR, cm −1 ): 3104 (ν NHtriazole), 1618 (ν -N=CH-), 1274 (ν C=S) 1055 (ν C-Br) 1 H NMR (500 MHz, DMSO-d6, δ/ppm): 14.18 (s, 1H, NH), 9.52 (s, 1H, -N=CH-), 7.97–8.06 (d, 2H, ArH), 7.92 (s, 1H, ArH), 7.77 (d, 1H, ArH), 7.59 (d, 1H, ArH), 7.47-7.54 (m, 4H, ArH), 2.41 (s, 3H, CH3) 13 C NMR (125 MHz, DMSO-d6, δ/ppm): 170.12 (C=S), 159.15 (C), 157.66 (CH=N), 153.81 (C), 151.07 (C), 143.96 (C), 135.16 (C), 134.51 (C), 131.21 (CH), 130.93 (2CH), 130.29 (CH), 129.29 (2CH), 128.94 (CH), 128.68 (C), 127.36 (CH), 127.14 (CH), 15.92 (CH3) MS (EI, 70 eV) m/z (%): 457 (M + 1).

3.2. Antimicrobial Activity

Results obtained by measuring the diameters of growth inhibition zones of the tested microorganisms, compared to ciprofloxacin and fluconazole, used as standard reference drugs, are presented in Table 1.

MIC, MBC, and MFC values of the new compound are presented in Tables 2 and 3. The results showed that MIC values ranged from 1.95 (Listeria monocytogenes) to 62.5 μg/mL, MBC values were between 3.9 and 125 μg/mL, and MFC scores ranged between 62.5 and 125 μg/mL.

3.3. In Vitro Antioxidant Capacity

The antioxidant capacity of the SB was determined by the DPPH bleaching method, and BHT and trolox were used as positive controls. The results are displayed in Table 4. The new compound showed a very low IC50 value (16.10 μg/mL), similar to that of BHT (16.39 μg/mL).

3.4. Cell Viability

SB did not lead to significant changes in HUVEC viability for doses lower than 0.1 μg/mL (Figure 1). Higher concentrations led to a dose-dependent viability decrease, compared with control.

3.5. Assessment of the Ability of the SB to Modulate Inflammatory Response and Oxidative Stress on HUVECs

Lipid peroxidation level (MDA), the ability to modulate inflammatory response (TNF-α, COX2), and the activity of enzymes involved in the prooxidant/antioxidant equilibrium (SOD1, NOS2) were appreciated. The ability of the SB to modulate oxidative stress was tested in vitro on HUVECs, using a glucose-enriched medium [36–38]. A SB concentration of 0.001 μg/mL was used for all experiments.

The effect of the newly synthetized compound on lipid peroxidation (MDA level) was assessed. SB administration decreased the MDA level compared with both control and glucose-enriched medium, thus reducing the lipid peroxidation in endothelial cells (Figure 2).

The TNF-α level was quantified through ELISA for the same SB concentration (Figure 3). Glucose-enriched medium slightly increased the TNF-α level. SB also increased the TNF-α level both alone and in combination with glucose.

The same SB concentration (0.001 μg/mL) was used to further test its effect on the protein level of the enzymes involved in the oxidant/antioxidant equilibrium and in the inflammatory response (SOD1, NOS2, and COX2).

An inflammatory marker (COX2) and antioxidant enzyme (constitutive SOD1 and inducible NOS2) expression was quantified by Western Blot (Figure 4).

COX2, an inflammatory marker, significantly decreased after both glucose and SB treatments, compared to control. Combined exposure (SB+G) strongly decreased the protein level of COX2 (Figure 4(b)). This finding is consistent with MDA levels and may be due to the antioxidant effect of the SB in this experimental setting. Interestingly, it is not consistent with TNF-α, a fact which might be explained by a different mechanism than oxidative stress that triggers an increase of TNF-α. Exposure to glucose-enriched medium significantly decreased SOD1. SB slightly decreased SOD1 activity compared with the control group, but SOD1 activity was maintained at a significantly higher level, compared with glucose ( ). Combination (SB+G) treatment significantly decreased SOD1 compared with both glucose and control (Figure 4(c)). Exposure to glucose increased significantly NOS2. The SB drastically decreased the NOS2 level compared with both the control and glucose groups (Figure 4(d)).

Correlation analysis, using Spearman’s coefficient for rank correlation (Table 5), revealed statistically significant positive correlations between MDA and enzyme (COX2, SOD1, and NOS2) levels. On the other hand, the TNF-α level negatively correlates with both MDA and all enzymes measured.

Cell morphology does not seem to be affected by exposure to the Schiff base compared to control. When exposed to high-glucose concentration, cells had a tendency to conglomerate and to form multilayered spherical bodies, with alteration of the actin filament disposition. The aspect of the cells receiving combination treatment was similar to those of controls (Figure 5).

4. Discussion

The structure of the Schiff base was established by elemental analysis and on the basis of its mass spectrum (MS), infrared spectrum (IR), and nuclear magnetic resonance ( 1 H-NMR and 13 C-NMR) spectra. The results of the C, H, N, S quantitative elemental analysis were in agreement with the calculated values, within ±0.4% of the theoretical values. The spectral data confirmed the formation of the SB. The recorded mass spectrum revealed the correct molecular ion peak (

), as suggested by the molecular formula. The absence of the NH2 asymmetric and symmetric stretching vibrations at 3281 cm −1 and 3186 cm −1 , and the presence of N=CH stretch absorption bands at 1618 cm −1 in the IR spectrum of the final compound provided strong evidences for the formation of the SB. The 1 H-NMR spectrum of the starting compound was recorded a signal characteristic for the amino protons, as a singlet, at 5.73 ppm. The absence of this signal from the 1 H-NMR spectrum of the newly synthesized compound and the presence of a singlet characteristic to the N=CH proton at 9.52 ppm further confirmed the condensation between the 4-amino-5-(4-methyl-2-phenylthiazol-5-yl)-4H-1,2,4-triazole-3-thiol and the 3-bromo-phenyl-carbaldehyde. The 13 C-NMR spectrum of the newly synthesized compound was consistent with the proposed structure.

The aim of the present study was to evaluate the antibacterial and antifungal activity of a new SB as well as its ability to modulate oxidative stress.

The new thiazolyl SB exerted moderate to good antibacterial activity against tested strains (Tables 1–3). The inhibition of bacterial growth was more pronounced in Gram-negative bacteria, especially in Pseudomonas aeruginosa strain, where the SB showed better activity compared with ciprofloxacin, used as the reference drug. Regarding antifungal activity, the compound showed a better anti-Candida effect than fluconazole, used as the reference drug. Previous studies showed that SBs have the ability to modulate oxidative stress [17, 39]. This ability can be exploited in order to use them as antibacterial drugs and/or as potential oxidative stress modulators in medicine. The SB was tested on endothelial cells exposed to a glucose-enriched environment.

High-carbohydrate intake, impaired glucose tolerance, and diabetes mellitus lead to hyperglycemia and chronic inflammatory status. Endothelial lesions are often involved in the pathology of these conditions [40]. During inflammatory episodes, such as response to injury, nitric oxide (NO) is released in order to modulate vascular tone. Since glycocalyx plays an important role in transducing the fluid stress to the cytoskeleton of the endothelial cells, vasodilator substance production is stimulated [40–42]. High-glucose concentration increases oxidative stress and influences the structure of the cytoskeleton. Exposure to high-glucose hyperosmolar medium induces, using an AQP1-dependent mechanism, remodeling of the F-actin and cytoskeleton [43]. Our results are consistent with these findings (Figure 5). A high-glucose level led to mitochondrial dysfunction and increased production of ROS [44, 45].

A glucose-enriched environment also triggers the release of proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-α), by the cells involved in immune reactions [46, 47], along with other proinflammatory molecules, such as CRP, interleukin 6, intercellular adhesion molecule 1, and VCAM-1. In diabetic patients, TNF-α was related with an atherogenic profile and with vascular complications [48]. A similar effect was obtained in our study, where higher levels of TNF-α were observed after hyperglycemia exposure. This effect was also seen after SB treatment and was augmented by the combined SB and high-glucose concentration. However, TNF-α production was negatively correlated with MDA and antioxidant enzymes (Table 5). This suggests that the increased TNF-α was not produced through enhanced oxidative stress, but through a different mechanism. Its clarification requires further studies. Since TNF-α acts as a promoter of leucocyte adhesion to the endothelium, the SB might be beneficial as antimicrobial, local immune response, and oxidative status modulator in the treatment of infectious diseases.

The results obtained by the DPPH study showed that the SB exhibited antioxidant activity. The low IC50 value, similar to the positive control (BHT), reflects a strong antioxidant activity in vitro. The new compound showed radical scavenging activity according to the DPPH method, the presence of the -SH group being probably responsible of the radical scavenging activity [49–51]. The effect of the SB on the oxidative stress was also tested in vitro on cell cultures (HUVECs), by assessing the MDA level, a marker of lipid peroxidation and the expression of two enzymes involved in the oxidative equilibrium (SOD1 and NOS2). The results showed that, at the tested concentration (0.001 μg/mL), SB decreased lipid peroxidation (MDA) and the protein level of certain enzymes involved in the modulation of oxidative stress and inflammatory response (COX2 and NOS2). These changes are consistent with the DPPH result and suggest an anti-inflammatory effect of the tested SB, mostly by interfering with the prooxidant mediators.

The ability of the SB, in low concentrations, to decrease lipid peroxidation, might be explained by its capacity to form complexes with the bivalent and trivalent metal ions located in the active center of the enzymes involved in the onset of the oxidative stress or in the scavenging of the prooxidant molecules [52–57]. The antioxidant effect on the human cells (Figures 2 and 4) is also consistent with the absence of morphological changes of the cells observed in the present study (Figure 5).

Considering antibacterial activity, especially against Pseudomonas aeruginosa, the decrease of the NOS2 protein level in HUVECs after SB exposure, it might be possible that the synthesis of NO by bacteria could also be reduced. One of the many proposed roles of NO in bacteria is to help protect the bacteria from host cell antibiotic-induced oxidative stress therefore, the inhibition of bacterial nitric oxide synthase has been identified as a promising antibacterial strategy, especially for resistant bacteria [58].

Nitric oxide synthase (NOS) inhibitor NO-donating drugs were reported to inhibit IL-1β production, modulate PGE2 production, and protect against apoptosis in human endothelial cells and human monocytes [59]. In type 2 diabetes, hyperglycemia stimulates endothelial cell migration in the retina, leading to retina neoangiogenesis and visual impairment by CXC receptor-4 stimulation and activation of the PI3K/Akt/eNOS signaling pathway. Therefore, SB modulation of the NOS2 might be beneficial for the endothelial dysfunction in hyperglycemia [60, 61].

Recent studies showed that the antibacterial and antifungal activity in general and antibiofilm activity of some newly identified classes seem to correlate with their ability to induce ROS synthesis [5]. The SB showed an anti-Candida effect, with a twofold increased activity compared with the consecrated antifungal fluconazole (Tables 2 and 3). Also, the results showed that SB reduced the SOD1 level and increased the activity of the proinflammatory cytokine (TNF-α). The antifungal effect could also be explained by the ability of the tested SB to form complexes between the azomethine group and the metal from the active center of the enzymes and also by its capacity to induce ROS production, similar with some antifungal azoles (e.g., miconazole) [5].

Additional studies are needed in order to clarify the effect of such compounds as SB and their role as adjuvant antioxidant, antimicrobial, and local immune response modulators (TNF-α) in the treatment of infectious diseases.

5. Conclusions

The new Schiff base exhibited antibacterial effects on both Gram-positive and Gram-negative bacteria, as well as antifungal activity against Candida albicans. The results of the present study show that the new SB plays a role in the prooxidant/antioxidant equilibrium. In the tested dose, SB does not change endothelial cell morphology, has an antioxidant effect, as demonstrated by the DPPH test, decreased lipid peroxidation (MDA), and decreased the inducible NOS2 level. Therefore, it can be considered a potential candidate with promising antioxidant properties that may be used as an adjuvant therapy in diseases caused by excessive free radical production. The decrease in COX2 and NOS2 levels also might suggest an anti-inflammatory action. A possible mechanism for the antibacterial activity on Gram-negative bacilli could include the decrease of the bacterial NOS level and the formation of complexes with metals located in the active center of certain bacterial enzymes. Also, the SB might potentially act as an antifungal agent, through ROS production in fungal biofilm cells. Its clarification requires further studies.

Data Availability

The data used to support the findings of this study are included within the article.

Conflicts of Interest

The authors declare that there is no conflict of interest regarding the publication of this paper.

Authors’ Contributions

Cristian Cezar Login, Şoimiţa Suciu, Ioana Bâldea, and Brînduşa Tiperciuc conceived and planned the experimental design. Brînduşa Tiperciuc performed the chemical synthesis and the characterization of the compounds. Dan Cristian Vodnar performed the antibacterial and antifungal investigation. Daniela Benedec performed the in vitro assessment of the antioxidant activity. Ioana Bâldea performed the in vitro testing on cell cultures. Nicoleta Decea performed the biochemical assessment of some oxidative stress markers. Cristian Cezar Login and Ioana Bâldea performed the statistical analysis. Cristian Cezar Login, Ioana Bâldea, Brînduşa Tiperciuc, Daniela Benedec, and Şoimiţa Suciu analyzed the data and wrote the paper. Cristian Cezar Login, Brînduşa Tiperciuc, Ioana Bâldea, and Daniela Benedec equally contributed to this work.

Acknowledgments

This research was funded by “Iuliu Hațieganu” University of Medicine and Pharmacy Cluj-Napoca internal research grant No. 4944/23/08.03.2016 (Cristian Cezar Login).

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Copyright

Copyright © 2019 Cristian Cezar Login et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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