ACE2 Peptide Fragment Interaction with Different S1 Protein Sites.

We study the effect of the peptide QAKTFLDKFNHEAEDLFYQ on the kinetics of the SARS-CoV-2 spike protein S1 binding to angiotensin-converting enzyme 2 (ACE2), with the aim to characterize the interaction mechanism of the SARS-CoV2 virus with its host cell. This peptide corresponds to the sequence 24-42 of the ACE2 α1 domain, which marks the binding site for the S1 protein. The kinetics of S1-ACE2 complex formation was measured in the presence of various concentrations of the peptide using bio-layer interferometry. Formation of the S1-ACE2 complex was inhibited by the peptide in cases where it was preincubated with S1 protein before the binding experiment. The kinetic analysis of S1-ACE2 complex dissociation revealed that preincubation stabilized this complex, and this effect was dependent on the peptide concentration as well as the preincubation time. The results point to the formation of the ternary complex of S1 with ACE2 and the peptide. This is possible in the presence of another binding site for the S1 protein beside the receptor-binding domain for ACE2, which binds the peptide QAKTFLDKFNHEAEDLFYQ. Therefore, we conducted computational mapping of the S1 protein surface, revealing two additional binding sites located at some distance from the main receptor-binding domain on S1. We suggest the possibility to predict and test the short protein derived peptides for development of novel strategies in inhibiting virus infections. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10989-021-10324-7.

In Vitro Ligand Binding Kinetics Explains the Pharmacokinetics of [18F]FE-PE2I in Dopamine Transporter PET Imaging.

Two of the most popular positron emission tomography (PET) tracers, [11C]PE2I and [18F]FE-PE2I, used to quantify dopamine transporters (DAT), display dissimilar kinetic behavior in in vivo assays. This difference can be explained by comparing values of kinetic rate constants, which characterize interaction of these tracers with DAT sites in vitro. At the same time, this kinetic analysis showed that the overall binding mechanism is similar for these two tracers and includes a fast step of complex formation followed by a slow isomerization step of this complex. Comparison with previous PE2I data revealed that isomerization of the DAT complex with PE2I occurs three times faster than in the case of FE-PE2I, which leads to the slower onset of peak specific binding of the former tracer in the DAT-rich regions. Therefore, ligands with slower isomerization on-rate, including [18F]FE-PE2I, seem to be better tracers in vivo, and their properties can be predicted in vitro.

Allosteric Effect of Adenosine Triphosphate on Peptide Recognition by 3'5'-Cyclic Adenosine Monophosphate Dependent Protein Kinase Catalytic Subunits.

The allosteric influence of adenosine triphosphate (ATP) on the binding effectiveness of a series of peptide inhibitors with the catalytic subunit of 3'5'-cyclic adenosine monophosphate dependent protein kinase was investigated, and the dependence of this effect on peptide structure was analyzed. The allosteric effect was calculated as ratio of peptide binding effectiveness with the enzyme-ATP complex and with the free enzyme, quantified by the competitive inhibition of the enzyme in the presence of ATP excess, and by the enzyme-peptide complex denaturation assay, respectively It was found that the principle "better binding-stronger allostery" holds for interactions of the studied peptides with the enzyme, indicating that allostery and peptide binding with the free enzyme are governed by the same specificity pattern. This means that the allosteric regulation does not include new ligand-protein interactions, but changes the intensity (strength) of the interatomic forces that govern the complex formation in the case of each individual ligand. We propose that the allosteric regulation can be explained by the alteration of the intrinsic dynamics of the protein by ligand binding, and that this phenomenon, in turn, modulates the ligand off-rate from its binding site as well as the binding affinity. The positive allostery could therefore be induced by a reduction in the enzyme's overall intrinsic dynamics.

Different States of Acrylodan-Labeled 3'5'-Cyclic Adenosine Monophosphate Dependent Protein Kinase Catalytic Subunits in Denaturant Solutions.

Fluorescence spectroscopy was used to differentiate between different states of acrylodan-labeled cAMP-dependent protein kinase catalytic subunits in urea, guanidine hydrochloride and 3-(N-morpholino)propanesulfonic acid solutions, by measuring changes in the emission spectrum of the protein-coupled dye, which is very sensitive to its microenvironment. Decomposition of the observed fluorescence spectra by a parameterized log-normal distribution function allowed the resolution of overlapping spectral bands and revealed the formation of three distinct protein states, denominated as native, denatured and unfolded structures. At low denaturant concentrations the formation of the denatured form from the native protein was observed, and this process was characterized by a blue-shift of the fluorescence spectrum of acrylodan, indicating that the dye was transferred into some water-deficit hydrophobic environment inside the protein molecule. Therefore, formation of a "dry molten globule" structure could be suggested in state. At high denaturant concentrations a red-shift of the emission spectrum of the protein-coupled probe was observed indicating significant extrusion of the dye molecule into water environment as a result of the unfolding of the protein structure.

Computational modeling of acrylodan-labeled cAMP dependent protein kinase catalytic subunit unfolding.

Structure of the cAMP-dependent protein kinase catalytic subunit, where the asparagine residue 326 was replaced with acrylodan-cystein conjugate to implement this fluorescence reporter group into the enzyme, was modeled by molecular dynamics (MD) method and the positioning of the dye molecule in protein structure was characterized at temperatures 300K, 500K and 700K. It was found that the acrylodan moiety, which fluorescence is very sensitive to solvating properties of its microenvironment, was located on the surface of the native protein at 300K that enabled its partial solvation with water. At high temperatures the protein structure significantly changed, as the secondary and tertiary structure elements were unfolded and these changes were sensitively reflected in positioning of the dye molecule. At 700K complete unfolding of the protein occurred and the reporter group was entirely expelled into water. However, at 500K an intermediate of the protein unfolding process was formed, where the fluorescence reporter group was directed towards the protein interior and buried in the core of the formed molten globule state. This different positioning of the reporter group was in agreement with the two different shifts of emission spectrum of the covalently bound acrylodan, observed in the unfolding process of the protein.

NCAM-deficient mice show prominent abnormalities in serotonergic and BDNF systems in brain - Restoration by chronic amitriptyline.

Mood disorders are associated with alterations in serotonergic system, deficient BDNF (brain-derived neurotrophic factor) signaling and abnormal synaptic plasticity. Increased degradation and reduced functions of NCAM (neural cell adhesion molecule) have recently been associated with depression and NCAM deficient mice show depression-related behavior and impaired learning. The aim of the present study was to investigate potential changes in serotonergic and BDNF systems in NCAM knock-out mice. Serotonergic nerve fiber density and SERT (serotonin transporter) protein levels were robustly reduced in the hippocampus, prefrontal cortex and basolateral amygdala of adult NCAM(-)(/-) mice. This SERT reduction was already evident during early postnatal development. [(3)H]MADAM binding experiments further demonstrated reduced availability of SERT in cell membranes of NCAM(-)(/-) mice. Moreover, the levels of serotonin and its major metabolite 5-HIAA were down regulated in the brains of NCAM(-)(/-) mice. NCAM(-)(/-) mice also showed a dramatic reduction in the BDNF protein levels in the hippocampus and prefrontal cortex. This BDNF deficiency was associated with reduced phosphorylation of its receptor TrkB. Importantly, chronic administration of antidepressant amitriptyline partially or completely restored these changes in serotonergic and BDNF systems, respectively. In conclusion, NCAM deficiency lead to prominent and persistent abnormalities in brain serotonergic and BDNF systems, which likely contributes to the behavioral and neurobiological phenotype of NCAM(-/-) mice.

Thermal Stability of Dopamine Transporters.

The thermal stabilities of the rat and mouse dopamine transporter (DAT) proteins were studied within the temperature range of 0-37°C. The inactivation of the protein was followed by monitoring changes in radioligand-specific binding. We found that the process followed a rate equation with first-order kinetics and was characterized by having a single rate constant k inact. The activation energies (E a) that were calculated from the Arrhenius plots (ln k inact vs. 1/T) were 43 ± 5 and 45 ± 6 kJ/mol for the rat (rDAT) and mouse (mDAT) transporters, respectively, and 44 ± 7 kJ/mol for rDAT from PC-6.3 cell line. These E a values were similar to the E a values of thermal inactivation of the muscarinic receptor from rat brain cortex and to the thermal inactivation of other transmembrane proteins. However, all of these activation energy values were significantly lower than the E a values for soluble single-subunit proteins of similar size. These results therefore suggest that the thermal stability of transmembrane proteins may be governed to a significant extent by cell membrane properties and by interactions between the membrane components and the protein. In contrast, the stability of soluble proteins seems to be mostly governed by protein structure and size, which determine the sum of the stabilizing intramolecular interactions within the protein molecule. It is therefore not surprising that cell membrane properties and composition may have significant effects on the functional properties of transmembrane proteins.

Thermodynamic aspects of cAMP dependent protein kinase catalytic subunit allostery.

Kinetics of thermal inactivation of acrylodan-labeled cAMP dependent protein kinase catalytic subunit, its binary complexes with ATP and peptide inhibitor PKI[5-24], respectively, and the ternary complex involving both of these ligands were studied at different temperatures (5-50 °C). The thermodynamic parameters ΔH and ΔS for ligand binding equilibria as well as for the allosteric interaction between the binding sites of these ligands were obtained by using the Van't Hoff analysis. The results indicated that more inter- and intra-molecular non-covalent bonds were involved in ATP binding with the protein when compared to the peptide binding. Similarly, nucleotide and peptide binding steps were accompanied with different entropy effects, while almost no entropy change accompanied PKI[5-24] binding, suggesting that the protein flexibility was not affected in this case. Differently from the binary complex formation the ternary complex formation was accompanied by a significant entropy change and with intensive formation of new non-covalent interactions (ΔH). At the same time both ligand binding steps as well as the allosteric interaction between ligand binding sites could be described by a common entropy-enthalpy compensation plot, pointing to a similar mechanism of these phenomena. It was concluded that numerous weak interactions govern the allostery of cAMP dependent protein kinase catalytic subunit.

Computational simulation of ligand docking to L-type pyruvate kinase subunit.

Computational blind docking approach was used for mapping of possible binding sites in L-type pyruvate kinase subunit for peptides, RRASVA and the phosphorylated derivative RRAS(Pi)VA, which model the phosphorylatable N-terminal regulatory domain of the enzyme. In parallel, the same docking analysis was done for both substrates of this enzyme, phosphoenolpyruvate (PEP) and adenosine diphosphate (ADP), and for docking of fructose 1,6-bisphosphate (FBP), which is the allosteric activator of the enzyme. The binding properties of the entire surface of the protein were scanned and several possible binding sites were identified in domains A and C of the protein, while domain B revealed no docking sites for peptides or for substrates or the allosteric regulator. It was found that the docking sites of different ligands were partially overlapping, pointing to the possibility that some regulatory effects, observed in the case of L-type pyruvate kinase, may be caused by the competition of different ligands for the same binding sites.

Sonication effects on non-radical reactions. A sonochemistry beyond the cavitation?

The kinetics of pH-independent hydrolysis of 4-methoxyphenyl dichloroacetate were investigated under ultrasonic irradiation with an application of 10% of the maximum power of the equipment and without sonication in acetonitrile-water binary mixtures with a content of acetonitrile ranging from 0.008 to 35 wt.%. Similar kinetic investigations were performed at intensities corresponding to 10%, 20%, 30%, 40%, and 50% of the input energy in solvent mixtures containing 10 wt.% and 25 wt.% acetonitrile. In parallel, the responses of KI and terephthalic acid dosimeters at applied irradiation levels were registered under the same experimental conditions. Significant kinetic sonication effects were found at sound intensities presumably not inducing cavitation in the solution. This result provides an experimental evidence of kinetic effects of ultrasound in the absence of cavitation. A disturbing impact of cavitation on the ultrasonic acceleration of the reaction was found. The implications of these findings were discussed.

Kinetics of acrylodan-labelled cAMP-dependent protein kinase catalytic subunit denaturation.

Fluorescence spectroscopy was used to study denaturation of cAMP-dependent protein kinase catalytic subunit labeled with an acrylodan moiety. The dye was covalently bound to a cystein residue introduced into the enzyme by replacement of arginine in position 326 in the native sequence, located near the enzyme active center. This labeling had no effect on catalytic activity of the enzyme, but provided possibility to monitor changes in protein structure through measuring the fluorescence spectrum of the dye, which is sensitive to changes in its environment. This method was used to monitor denaturation of the protein kinase catalytic subunit and study the kinetics of this process as well as influence of specific ligands on stability of the protein. Stabilization of the enzyme structure was observed in the presence of adenosine triphosphate, peptide substrate RRYSV and inhibitor peptide PKI[5-24].

Computer modeling of the dynamic properties of the cAMP-dependent protein kinase catalytic subunit.

The structural dynamics of the cAMP-dependent protein kinase catalytic subunit were modeled using molecular dynamics computational methods. It was shown that the structure of this protein as well as its complexes with ATP and peptide ligand PKI(5-24) consisted of a large number of rapidly inter-converting conformations which could be grouped into subsets proceeding from their similarity. This cluster analysis revealed that conformations which correspond to the "opened" and "closed" structures of the protein were already present in the free enzyme, and most surprisingly co-existed in enzyme-ATP and enzyme-PKI(5-24) complexes as well as in the ternary complex, which included both of these ligands. The results also demonstrated that the most mobile structure segments of the protein were located in the regions of substrate binding sites and that their dynamics were most significantly affected by the binding of the ATP and peptide ligand.

Kinetic sonication effects in aqueous acetonitrile solutions. Reaction rate levelling by ultrasound.

The kinetics of the pH-independent hydrolysis of 4-methoxyphenyl dichloroacetate were investigated with and without ultrasonic irradiation in acetonitrile-water binary mixtures containing 0.008 to 35 wt.% of acetonitrile and the kinetic sonication effects (kson/knon) were calculated. Molecular dynamics (MD) simulations of the structure of the solutions were performed with ethyl acetate as the model ester. The ester is preferentially solvated by acetonitrile. The excess of acetonitrile over water in the solvation shell grows fast with an increase in the co-solvent content in the bulk solution. In parallel, the formation of a second solvation shell rich in acetonitrile takes place. Significant kinetic sonication effects for the hydrolysis were explained with facile destruction of the diffuse second solvation shell followed by a rearrangement of the remaining solvent layer under sonication. The rate levelling effect of ultrasound was discussed. In an aqueous-organic binary solvent, independent of the solvent composition, the ultrasonic irradiation evokes changes in the reaction medium which result in an almost identical solvation state of the reagent thus leading to the reaction rate levelling.

Interaction of non-phosphorylated liver pyruvate kinase with fructose 1,6-bisphosphate and peptides that mimic the phosphorylatable N-terminus of the enzyme.

The interaction of non-phosphorylated L-type pyruvate kinase (L-PK) with fructose 1,6-bisphosphate (FBP), which is an allosteric activator of the phosphorylated enzyme, and peptides that mimic the phosphorylatable N-terminal regulatory domain of the enzyme, was studied. It was found that the catalytic activity of the enzyme was not enhanced in the presence of FBP, and this ligand acted as a relatively weak reversible inhibitor of the enzyme activity in the micromolar concentration range. The phosphorylation site analogue peptides RRASVA and RRAAVA had no effect on the activity of the enzyme, while the phosphorylated peptide RRAS(Pi)VA reversibly inhibited the enzyme and this process was characterised by the Ki value 47 µM. As the phosphorylated form of L-PK is a subject of significant allosteric regulation by FBP, it was concluded that phosphorylation should function as a molecular switch of the allosteric properties of this enzyme.

Novel systemically active galanin receptor 2 ligands in depression-like behavior.

Neuropeptide galanin and its three G-protein coupled receptors, galanin receptor type 1-galanin receptor type 3 (GalR1-GalR3), are involved in the regulation of numerous physiological and disease processes, and thus represent tremendous potential in neuroscience research and novel drug lead development. One of the areas where galanin is involved is depression. Previous studies have suggested that activation of GalR2 leads to attenuation of depression-like behavior. Unfortunately, lack of in vivo usable subtype specific ligands hinders testing the role of galanin in depression mechanisms. In this article, we utilize an approach of increasing in vivo usability of peptide-based ligands, acting upon CNS. Thus, we have synthesized a series of novel systemically active galanin analogs, with modest preferential binding toward GalR2. We have shown that specific chemical modifications to the galanin backbone increase brain levels upon i.v. injection of the peptides. Several of the new peptides, similar to a common clinically used antidepressant medication imipramine, exerted antidepressant-like effect in forced swim test, a mouse model of depression, at a surprisingly low dose range (< 0.5 mg/kg). We chose one of the peptides, J18, for more thorough study, and showed its efficacy also in another mouse depression model (tail suspension test), and demonstrated that its antidepressant-like effect upon i.v. administration can be blocked by i.c.v. galanin receptor antagonist M35. The effect of the J18 was also abolished in GalR2KO animals. All this suggests that systemically administered peptide analog J18 exerts its biological effect through activation of GalR2 in the brain. The novel galanin analogs represent potential drug leads and a novel pharmaceutical intervention for depression.

Kinetic sonication effects in light of molecular dynamics simulation of the reaction medium.

Molecular dynamics (MD) simulation of the structure of ethyl acetate solutions in two water-ethanol mixtures was performed at 280 and 330K. The MD simulations revealed that ethyl acetate was preferentially solvated by ethanol, water being mainly located in the next solvation layer. With increasing temperature ethanol was gradually replaced by water in the first solvation shell. These findings explain the decrease in the rate of ester hydrolysis with increasing molar ratio of ethanol in the solution as the reaction rate was linearly dependent on the relative ethanol content in the first solvation shell of the ester. Predominance of ethanol results in decreased polarity and water activity in the shell and accordingly in a decreased reaction rate. Based on the results of the MD simulations, the principal conclusion of this work is that ultrasound enhances the kinetic energy (the effective temperature) of species in the solution and, in this way, evokes shifts in the solvation equilibria thus affecting the reaction rate. It appears that ultrasound does not completely break down the solvent shells or clusters in the solution as previously believed. Phenomena of thermo-solvatochromism and reaction rate levelling by ultrasound in binary solvents are described.

Novel galanin receptor subtype specific ligand in depression like behavior.

Neuropeptide galanin and its three receptors, galanin receptor type 1-galanin receptor type 3, are known to be involved in the regulation of numerous psychological processes, including depression. Studies have suggested that stimulation of galanin receptor type 2 (GalR2) leads to attenuation of the depression-like behavior in animals. However, due to the lack of highly selective galanin subtype specific ligands the involvement of different receptors in depression-like behavior is yet not fully known. In the present study we introduce a novel GalR2 selective agonist and demonstrate its ability to produce actions consistent with theorized GalR2 functions and analogous to that of the anti-depressant, imipramine.

Probing L-pyruvate kinase regulatory phosphorylation site by mutagenesis.

The activity of L-type pyruvate kinase (L-PK, ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) is regulated by phosphorylation of serine residue 12 of the N-terminal regulatory domain MEGPAGYLRR(10)AS ( 12 )VAQLTQEL(20)GTAFF of the protein. In this report we studied the effect of the point mutations around this phosphorylation site on the catalytic properties of this enzyme, by introducing amino acids A, L, K, Q and E into positions 9, 10 and 13 of this peptide sequence. It was found that some of these mutations in positions 9 and 10, although occurring at great distances from the enzyme's active site, affected the enzyme's activity by decreasing the effectiveness of phosphoenolpyruvate binding (PEP) with the enzyme, but had practically no influence on the binding effectiveness of the second substrate ADP. A similar asymmetric effect on the binding of these substrates was previously observed after phosphorylation of the enzyme regulatory N-domain peptide, and also after proteolytic truncation of the same N-terminal part of L-PK. All these results could be explained by the internal complex formation between the N-domain peptide and the enzyme's main body. The present study delineated the specificity of the internal binding site and revealed the possibility that the regulatory effect could be modulated by selecting mutation sites and amino acids introduced into the N-terminal domain structure.

Effect of two simultaneous aza-ß3-amino acid substitutions on recognition of peptide substrates by cAMP dependent protein kinase catalytic subunit.

Peptidomimetic analogs of the hexapeptide RRASVA, containing simultaneously two aza-ß(3)-amino acid residues in different positions of this sequence, except for the phosphorylatable serine residue, were synthesized and tested as substrates for the cAMP-dependent protein kinase catalytic subunit. All these peptidomimetics were phosphorylated by the enzyme and this reaction was characterized by the K(m) and k(cat) values as well as by the second-order rate constants k(II). Affinity and reactivity of all peptidomimetics was lower than that for the parent peptide RRASVA. The effect of backbone modification was dependent upon the positions where these two aza-ß(3) residues were located, although the sequence of amino acid side groups remained the same in all compounds. It was found that the influence of two backbone modifications in the substrate structure was not described additively, i.e. the effect of each structural alteration was dependent upon the position of the second modification. The results were in agreement with the concept of specificity-determining clusters in the sequence of peptide and peptidomimetic ligands, which predominantly determine the molecular recognition of these ligands by their target sites and therefore serve as major modification points for the design of activity of peptidomimetic ligands.

Novel galanin receptor subtype specific ligands in feeding regulation.

Galanin a 29/30-residue neuropeptide has been implicated in several functions in the central nervous system, including the regulation of food consumption. Galanin and its analogues administered intraventricularly or into the hypothalamic region of brain have been shown to reliably and robustly stimulate the consumption of food in sated rodents. Three galanin receptor subtypes have been isolated, all present in the hypothalamus, but little is known about their specific role in mediating this acute feeding response. Presently, we introduce several novel GalR2 selective agonists and then compare the most selective of these novel GalR2 subtype selective agonists to known GalR1 selective agonist M617 for their ability to stimulate acute consumption of several foods shown to be stimulated by central administration of galanin. GalR1 selective agonist M617 markedly stimulated acute consumption of high-fat milk, but neither GalR2 selective agonist affected either high-fat milk or cookie mash intake. The present results are consistent with the involvement of GalR1 in mediating the acute feeding consumption by galanin and suggest an approach applicable to exploring galanin receptor specificity in normal and abnormal behavior and physiology.