Distinctive Nucleic Acid Recognition by Lysine-Embedded Phenanthridine Peptides.

Three new phenanthridine peptide derivatives (19, 22, and 23) were synthesized to explore their potential as spectrophotometric probes for DNA and RNA. UV/Vis and circular dichroism (CD) spectra, mass spectroscopy, and computational analysis confirmed the presence of intramolecular interactions in all three compounds. Computational analysis revealed that compounds alternate between bent and open conformations, highlighting the latter's crucial influence on successful polynucleotide recognition. Substituting one glycine with lysine in two regioisomers (22, 23) resulted in stronger binding interactions with DNA and RNA than for a compound containing two glycines (19), thus emphasizing the importance of lysine. The regioisomer with lysine closer to the phenanthridine ring (23) exhibited a dual and selective fluorimetric response with non-alternating AT and ATT polynucleotides and induction of triplex formation from the AT duplex. The best binding constant (K) with a value of 2.5 × 107 M-1 was obtained for the interaction with AT and ATT polynucleotides. Furthermore, apart from distinguishing between different types of ds-DNA and ds-RNA, the same compound could recognize GC-rich DNA through distinct induced CD signals.

Sulfone-based human liver pyruvate kinase inhibitors - Design, synthesis and in vitro bioactivity.

Non-alcoholic fatty liver disease (NAFLD) is a prevalent pathological condition characterised by the accumulation of fat in the liver. Almost one-third of the global population is affected by NAFLD, making it a significant health concern. However, despite its prevalence, there is currently no approved drug specifically designed for the treatment of NAFLD. To address this critical gap, researchers have been investigating potential targets for NAFLD drug development. One promising candidate is the liver isoform of pyruvate kinase (PKL). In recent studies, Urolithin C, an allosteric inhibitor of PKL, has emerged as a potential lead compound for therapeutic intervention. Building upon this knowledge, our team has conducted a comprehensive structure-activity relationship of Urolithin C. In this work, we have employed a scaffold-hopping approach, modifying the urolithin structure by replacing the urolithin carbonyl with a sulfone moiety. Our structure-activity relationship analysis has identified the sulfone group as particularly favourable for potent PKL inhibition. Additionally, we have found that the presence of catechol moieties on the two aromatic rings further improves the inhibitory activity. The most promising inhibitor from this new series displayed nanomolar inhibition, boasting an IC50 value of 0.07 µM. This level of potency rivals that of urolithin D and significantly surpasses the effectiveness of urolithin C by an order of magnitude. To better understand the molecular interactions underlying this inhibition, we obtained the crystal structure of one of the inhibitors complexed with PKL. This structural insight served as a valuable reference point, aiding us in the design of inhibitors.

Membrane Separation of Chicken Byproduct Hydrolysate for Up-Concentration of Bioactive Peptides.

Membrane processes, such as microfiltration, ultrafiltration, and nanofiltration, are increasingly used for various applications in both upstream and downstream processing. Membrane-based processes play a critical role in the field of separation/purification of biotechnological products, including protein production/purification. The possibility of using membranes to separate peptides from a chicken byproduct hydrolysate and the effect of the performed downstream processing on the DPP-IV dipeptidyl peptidase IV (DPP-IV) inhibitory activity of mechanical deboning chicken residue (MDCR) has been investigated. The chicken byproduct hydrolysate was prepared by enzymatic hydrolysis followed by microfiltration (MF), ultrafiltration (UF), nanofiltration (NF), and reverse osmosis (RO) separation. Comparing all separation treatments, hydrolysates processed only by MF and UF show the best DPP-IV inhibition (59.5-60.0% at 1 mg/mL and 34.2-40.7% at 0.5 mg/mL). These samples show dose-responsive behavior. Bioactivity was correlated with molecular weight distribution profiles and average molecular weights. The nanofiltration process notably decrease the inhibitory activity, and these permeates show low DPP-IV inhibition (9.5-21.8% at 1 mg/mL and 3.6-12.1% at 0.5 mg/mL). The size-exclusion chromatography-organic carbon detection-organic nitrogen detection (LC-OCD-OND) analysis confirms that NF and RO would retain the bioactive peptides in the concentrate in comparison to MF and UF. Bioactivity was correlated with molecular weight distribution profiles and average molecular weights. Permeates after ultrafiltration show an IC50 value of 0.75 mg/mL, comparable to other potent DPP-IV inhibitors derived from various food sources, and significantly more potent compared to the microfiltration sample, which shows an IC50 value of 1.04 mg/mL. The average molecular weight of the permeates calculated from the SEC chromatograms was 883 g/mol for UF and 1437 g/mol for MF. Of the four membranes studied, the UF membrane shows the best separation properties with respect to maximizing the yield and up-concentration of the bioactive peptides. Overall, UF was demonstrated to be a feasible technology for the removal of the undesired high-molecular-weight substances and up-concentration of small-molecular-weight bioactive peptides from chicken byproduct hydrolysate. These peptides might exhibit biological activity and could offer several health benefits. There is a high potential for the use of bioactive peptides, and more research in this field can lead to promising results that have significant effects in the food and medical industries.

The role of biospectroscopy and chemometrics as enabling technologies for upcycling of raw materials from the food industry.

It is important to utilize the entire animal in meat and fish production to ensure sustainability. Rest raw materials, such as bones, heads, trimmings, and skin, contain essential nutrients that can be transformed into high-value products. Enzymatic protein hydrolysis (EPH) is a bioprocess that can upcycle these materials to create valuable proteins and fats. This paper focuses on the role of spectroscopy and chemometrics in characterizing the quality of the resulting protein product and understanding how raw material quality and processing affect it. The article presents recent developments in chemical characterisation and process modelling, with a focus on rest raw materials from poultry and salmon production. Even if some of the technology is relatively mature and implemented in many laboratories and industries, there are still open challenges and research questions. The main challenges are related to the transition of technology and insights from laboratory to industrial scale, and the link between peptide composition and critical product quality attributes.

The Mechanism of Anti-Tumor Activity of 6-Morpholino- and 6-Amino-9-Sulfonylpurine Derivatives on Human Leukemia Cells.

The aim of this study was to explore the mechanism of antitumor effect of (E)-6-morpholino-9-(styrylsulfonyl)-9H-purine (6-Morpholino-SPD) and (E)-6-amino-9-(styrylsulfonyl)-9H-purine (6-Amino-SPD). The effects on apoptosis induction, mitochondrial potential, and accumulation of ROS in treated K562 cells were determined by flow cytometry. The RT-PCR method was used to measure the expression of Akt, CA IX, caspase 3, and cytochrome c genes, as well as selected miRNAs. Western blot analysis was used to determine the expression of Akt, cytochrome c, and caspase 3. The results demonstrate the potential of the tested derivatives as effective antitumor agents with apoptotic-inducing properties. In leukemic cells treated with 6-Amino-SPD, increased expression of caspase 3 and cytochrome c genes was observed, indicating involvement of the intrinsic mitochondrial pathway in the induction of apoptosis. Conversely, leukemic cells treated with 6-Morpholino-SPD showed reduced expression of these genes. The observed downregulation of miR-21 by 6-Morpholino-SPD may contribute to the induction of apoptosis and disruption of mitochondrial function. In addition, both derivatives exhibited increased expression of Akt and CA IX genes, suggesting activation of the Akt/HIF pathway. However, the exact mechanism and its relations to the observed overexpression of miR-210 need further investigation. The acceptable absorption and distribution properties predicted by ADMET analysis suggest favorable pharmacokinetic properties for these derivatives.

Phenanthridine-pyrene conjugates as fluorescent probes for DNA/RNA and an inactive mutant of dipeptidyl peptidase enzyme.

Two novel conjugate molecules were designed: pyrene and phenanthridine-amino acid units with a different linker length between the aromatic fragments. Molecular modelling combined with spectrophotometric experiments revealed that in neutral and acidic buffered water solutions conjugates predominantly exist in intramolecularly stacked conformations because of the π-π stacking interaction between pyrene and phenanthridine moieties. The investigated systems exhibited a pH-dependent excimer formation that is significantly red-shifted relative to the pyrene and phenanthridine fluorescence. While the conjugate with a short linker showed negligible spectrophotometric changes due to the polynucleotide addition, the conjugate with a longer and more flexible linker exhibited a micromolar and submicromolar binding affinity for ds-polynucleotides and inactivated a mutant of dipeptidyl peptidase enzyme E451A. Confocal microscopy revealed that the conjugate with the longer linker entered the HeLa cell membranes and blue fluorescence was visualized as the dye accumulated in the cell membrane.

Exploration of Novel Urolithin C Derivatives as Non-Competitive Inhibitors of Liver Pyruvate Kinase.

The inhibition of liver pyruvate kinase could be beneficial to halt or reverse non-alcoholic fatty liver disease (NAFLD), a progressive accumulation of fat in the liver that can lead eventually to cirrhosis. Recently, urolithin C has been reported as a new scaffold for the development of allosteric inhibitors of liver pyruvate kinase (PKL). In this work, a comprehensive structure-activity analysis of urolithin C was carried out. More than 50 analogues were synthesized and tested regarding the chemical features responsible for the desired activity. These data could pave the way to the development of more potent and selective PKL allosteric inhibitors.

Anthraquinone derivatives as ADP-competitive inhibitors of liver pyruvate kinase.

Liver pyruvate kinase (PKL) is a major regulator of metabolic flux and ATP production during liver cell glycolysis and is considered a potential drug target for the treatment of non-alcoholic fatty liver disease (NAFLD). In this study, we report the first ADP-competitive PKL inhibitors and identify several starting points for the further optimization of these inhibitors. Modeling and structural biology guided the optimization of a PKL-specific anthraquinone-based compound. A structure-activity relationship study of 47 novel synthetic derivatives revealed PKL inhibitors with half-maximal inhibitory concentration (IC50) values in the 200 nM range. Despite the difficulty involved in studying a binding site as exposed as the ADP site, these derivatives feature expanded structural diversity and chemical spaces that may be used to improve their inhibitory activities against PKL. The obtained results expand the knowledge of the structural requirements for interactions with the ADP-binding site of PKL.

6-Morpholino- and 6-amino-9-sulfonylpurine derivatives. Synthesis, computational analysis, and biological activity.

The synthesis of novel 6-chloro/morpholino/amino/-9-sulfonylpurine derivatives was accomplished in two ways, either (i) involving the condensation reaction of 6-chloropurine with commercially available arylsulfonyl chlorides in acetone and the presence of aqueous KOH at 0 °C, followed by the substitution of C6-chlorine with morpholine, or (ii) employing a reversed synthetic approach where 6-morpholinopurine and commercially available adenine bases were reacted with the corresponding alkyl, 2-arylethene and arylsulfonyl chlorides giving the N9 sulfonylated products, the latter particularly used where prior nonselective sulfonylation was observed. In both approaches, the sulfonylation reaction occurred regioselectively at the purine N9 position lacking any concurrent N7 derivatives, except in the case of a smaller methyl substituent on SO2 and the free amino group at C6 of the purine ring. The tautomeric features of initial N9 unsubstituted purines, as well as stability trends among the prepared N-9-sulfonylpurine derivates, were investigated using DFT calculations with an important conclusion that electron-donating C6 substituents are beneficial for the synthesis as they both promote the predominance of the desired N9 tautomers and help to assure the stability of the final products. The newly synthesized 6-morpholino and 6-amino-9-sulfonylpurine derivatives showed antiproliferative activity on human carcinoma, lymphoma, and leukemia cells. Among the tested compounds, 6-morpholino 17 and 6-amino 22 derivatives, with trans-ß-styrenesulfonyl group attached at the N9 position of purine, proved to be the most effective antiproliferative agents, causing accumulation of leukemia cells in subG0 cell cycle phase.

Flexibility and Preorganization of Fluorescent Nucleobase-Pyrene Conjugates Control DNA and RNA Recognition.

We synthesized a new amino acid-fluorescent nucleobase derivative (qAN1-AA) and from it two new fluorescent nucleobase-fluorophore (pyrene) conjugates, whereby only the analogue with the longer and more flexible linker (qAN1-pyr2) self-folded into intramolecularly stacked qAN1/pyrene conformation, yielding characteristic, 100 nm-red-shifted emission (λmax = 500 nm). On the contrary, the shorter and more rigid linker resulted in non-stacked conformation (qAN1-pyr1), characterized by the emission of free pyrene at λmax = 400 nm. Both fluorescent nucleobase-fluorophore (pyrene) conjugates strongly interacted with ds-DNA/RNA grooves with similar affinity but opposite fluorescence response (due to pre-organization), whereas the amino acid-fluorescent base derivative (qAN1-AA) was inactive. However, only intramolecularly self-folded qAN1-pyr2 showed strong fluorescence selectivity toward poly U (Watson-Crick complementary to qAN1 nucleobase) and poly A (reverse Hoogsteen complementary to qAN1 nucleobase), while an opposite emission change was observed for non-complementary poly G and poly C. Non-folded analogue (qAN1-pyr1) showed no ss-RNA selectivity, demonstrating the importance of nucleobase-fluorophore pre-organization.

DNA/RNA recognition controlled by the glycine linker and the guanidine moiety of phenanthridine peptides.

The binding of four phenanthridine-guanidine peptides to DNA/RNA was evaluated via spectrophotometric/microcalorimetric methods and computations. The minor structural modifications-the type of the guanidine group (pyrrole guanidine (GCP) and arginine) and the linker length (presence or absence of glycine)-greatly affected the conformation of compounds and consequently the binding to double- (ds-) and single-stranded (ss-) polynucleotides. GCP peptide with shorter linker was able to distinguish between RNA (A-helix) and DNA (B-helix) by different circular dichroism response at 295 nm and thus can be used as a chiral probe. Opposed to the dominant stretched conformation of GCP peptide with shorter linker, the more flexible and longer linker of its analogue enabled the molecule to adopt the intramolecularly stacked form which resulted in weaker yet selective binding to DNA. Beside efficient organization of ss-polynucleotide structures, GCP peptide with shorter linker bound stronger to ss-DNA/RNA compared to arginine peptides which emphasize the importance of GCP unit.

Antiproliferative and proapoptotic activity of molecular copper(II) complex of N-1-tosylcytosine.

In an attempt to enhance the previously observed antiproliferative capacity of 1-(p-toluenesulfonyl)cytosine (N-1-tosylcytosine, ligand 1), its copper(II) complex (Cu(1-TsC-N3)2Cl2, complex 2) was prepared and tested in vitro on various carcinoma and leukemia cells. The comparative in vitro studies using the ligand 1, the complex 2, CuCl2x2H2O salt (salt 3) and the 1:2 mixture of the salt 3 and ligand 1 (mixture 4) were performed on normal (WI38), human carcinoma (HeLa, CaCo2, MiaPaCa2, SW620), lymphoma (Raji) and leukemia (K562) cell lines. Significantly elevated concentration of the intracellular copper after treatment of K562 cells and HeLa cells during 2h with complex 2 (7.83 vs. 5.4 times) was detected by atomic absorption spectroscopy. Cytotoxicity was analyzed by MTT assay. We found that antiproliferative capacity of the tested compounds varies (IC50 after 72h of exposure: 0.6×10-6M to>100×10-6M). Leukemia and lymphoma cells were found the most sensitive to complex 2 which showed more than 100 times higher in vitro activity against K562 cells than ligand 1. Apoptotic morphological changes, an externalization of phosphatydilserine, and changes in the mitochondrial membrane potential of treated cells were found. The caspase-3 activity in HeLa and K562 cells was measured by caspase-3 colorimetric assay kit. Caspase-3 was not activated in the treated K562 cells while salt 3 and the mixture 4 in the HeLa cells significantly increased tested enzyme activity. These findings suggest that copper(II) in the molecular complex 2 by improving entry of the N-1-tosylcytosine 1 into cells increases its antiproliferative capacity. In summary, the present study demonstrated that complex 2 possesses an antileukemic effect on K562 cells, and its anticancer activity was attributed with induction of apoptosis. The exact mechanism of apoptosis induction by complex 2 must be further investigated.

Conjugates of Classical DNA/RNA Binder with Nucleobase: Chemical, Biochemical and Biomedical Applications.

Among the most intensively studied classes of small molecules (molecular weight < 650) in biomedical research are small molecules that non-covalently bind to DNA/RNA, and another intensively studied class is nucleobase derivatives. Both classes have been intensively elaborated in many books and reviews. However, conjugates consisting of DNA/RNA binder covalently linked to nucleobase are much less studied and have not been reviewed in the last two decades. Therefore, this review summarized reports on the design of classical DNA/RNA binder - nucleobase conjugates, as well as data about their interactions with various DNA or RNA targets, and even in some cases protein targets are involved. According to these data, the most important structural aspects of selective or even specific recognition between small molecule and target are proposed, and where possible related biochemical and biomedical aspects were discussed. The general conclusion is that this, rather new class of molecules showed an amazing set of recognition tools for numerous DNA or RNA targets in the last two decades, as well as few intriguing in vitro and in vivo selectivities. Several lead research lines show promising advancements toward either novel, highly selective markers or bioactive, potentially druggable molecules.

The transformation from 2°-amine to 3°-amine of cyclam ring alters the fragmentation patterns of 1-tosylcytosine-cyclam conjugates.

The novel N-1-sulfonylcytosine-cyclam conjugates 1 and 2 conjugates are ionized by electrospray ionization mass spectrometry (ESI MS) in positive and negative modes (ES+ and ES- ) as singly protonated/deprotonated species or as singly or doubly charged metal complexes. Their structure and fragmentation behavior is examined by collision induced experiments. It was observed that the structure of the conjugate dictated the mode of the ionization: 1 was analyzed in ES- mode while 2 in positive mode. Complexation with metal ions did not have the influence on the ionization mode. Zn2+ and Cu2+ complexes with ligand 1 followed the similar fragmentation pattern in negative ionization mode. The transformation from 2°-amine in 1 to 3°-amine of cyclam ring in 2 leads to the different fragmentation patterns due to the modification of the protonation priority which changed the fragmentation channels within the conjugate itself. Cu2+ ions formed complexes practically immediately, and the priority had the cyclam portion of the ligand 2. The structure of the formed Zn2+ complexes with ligand 2 depended on the number of 3° amines within the cyclam portion of the conjugate and the ratio of the metal:ligand used. The cleavage of the cyclam ring of metal complexes is driven by the formation of the fragment that suited the coordinating demand of the metal ions and the collision energy applied. Finally, it was shown that the structure of the cyclam conjugate dictates the fragmentation reactions and not the metal ions.

C5-Morpholinomethylation of N1-sulfonylcytosines by a one-pot microwave assisted Mannich reaction.

A fast and efficient route for the introduction of a methylene bridged-amine (morpholinomethyl) functionality in the C5 position of the sulfonylated cytosine nucleobase has been developed. First, novel N1-sulfonylcytosine derivatives 3-6 were prepared by the condensation of silylated cytosine with selected sulfonyl chlorides. They were subsequently transformed to 5-morpholinomethyl-N1-sulfonylcytosine derivatives (8, 12-15) using microwave irradiation. As a result of cytosine ring opening in N1-tosylcytosine, depending on the reaction conditions, peculiar tosyl-urea derivative 9 has been isolated, which provided additional insight into the reaction pathway. The influence of the C5-substituent on the antiproliferative activity has been evaluated by performing the MTT test on U251, MCF-7 and MOLT-4 tumor cell-lines.

ESI-MS studies of the non-covalent interactions between biologically important metal ions and N-sulfonylcytosine derivatives.

The aim of this report is to present the electrospray ionization mass spectrometry results of the non-covalent interaction of two biologically active ligands, N-1-(p-toluenesulfonyl)cytosine, 1-TsC, 1 and N-1-methanesulfonylcytosine, 1-MsC, 2 and their Cu(II) complexes Cu(1-TsC-N3)2 Cl2 , 3 and Cu(1-MsC-N3)2 Cl2 and 4 with biologically important cations: Na+ , K+ , Ca2+ , Mg2+ and Zn2+ . The formation of various complex metal ions was observed. The alkali metals Na+ and K+ formed clusters because of electrostatic interactions. Ca2+ and Mg2+ salts produced the tris ligand and mixed ligand complexes. The interaction of Zn2+ with 1-4 produced monometal and dimetal Zn2+ complexes as a result of the affinity of Zn2+ ions toward both O and N atoms. Copyright © 2016 John Wiley & Sons, Ltd.

Advances in Peptide-based DNA/RNA-Intercalators.

Interactions between DNA/RNA and huge variety of peptides are quite common in nature, controlling vast number of processes. Also, there are several naturally occurring small molecules which contain peptide and DNA intercalator in structure, whereby their biological activity is based on synergistic interactions of both components; for instance bis-intercalator echinomycin. Versatility of synthetic approaches to short peptides allowed their usage as simple recognition units within the DNA or RNA grooves or as backbone carriers for variety of bioactive substituents attached to amino acid side chains, one of very popular examples being PNAs. Such peptide-backbones were also used for synthesis of novel intercalators and poly-intercalators, many of them showing intriguing novel DNA/RNA interacting properties.