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.

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.

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.

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.

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.

Aza-beta(3)-amino acid containing peptidomimetics as cAMP-dependent protein kinase substrates.

Peptidomimetic analogs of the peptide RRASVA, known as the "minimal substrate" of the catalytic subunit of the cAMP-dependent protein kinase (PKA), were synthesized by consecutive replacement of natural amino acids by their aza-beta(3) analogs. The peptidomimetics were tested as PKA substrates and the kinetic parameters of the phosphorylation reaction were determined. It was found that the interaction of these peptidomimetics with the enzyme active center was sensitive to the location of the backbone modification, while the maximal rate of the reaction was practically not affected by the structure of substrates. The pattern of molecular recognition of peptidomimetics was in agreement with the results of structure modeling and also with the results of computational docking study of peptide and peptidomimetic substrates with the active center of PKA. It was concluded that the specificity determining factors which govern substrate recognition by the enzyme should be grouped along the phosphorylatable substrate, and such clustering might open new perspectives for pharmacophore design of peptides and peptide-like ligands.

Mechanism and stoichiometry of 2,2-diphenyl-1-picrylhydrazyl radical scavenging by glutathione and its novel alpha-glutamyl derivative.

Kinetic mechanism and stoichiometry of scavenging the 2,2-diphenyl-1-picrylhydrazyl radical by glutathione and its novel analog, containing alpha-glutamyl residue in place of the gamma-glutamyl moiety, were studied using different ratios of reagents. At low concentrations of the peptides, the process was described as a bimolecular reaction obeying the stoichiometric ratio 1:1. However, at excess of peptides the formation of a non-covalent complex between the reagents was discovered and characterized by dissociation constants K = 0.61 mM for glutathione and K = 0.27 mM for the glutathione alpha-glutamyl analog, respectively. The complex formation was followed by a reaction step that was characterized by the similar rate constant k = 0.02 s(-1) for both peptides. Thus, the apparently different antioxidant activity of these two peptides, observed under common assay conditions, was determined by differences in the formation of this non-covalent complex.

Kinetic mechanism of dopamine transporter interaction with 1-(2-(bis-(4-fluorophenyl)methoxy)ethyl)-4-(3-phenylpropyl)piperazine (GBR 12909).

Interaction of piperazine-based dopamine transporter inhibitor GBR12909 with rat dopamine transporters has been studied by means of competition kinetics analysis, employing [(3)H]PE2I as the reporter ligand. It has been found that GBR12909 is capable of inducing so-called "slow isomerization step" upon binding to DAT, probably consisting of a conformational change in the transporter protein. The mechanism exhibited by GBR12909 appears to be similar to the mechanism of PE2I that has been reported earlier and also confirms previous observations for GBR12783 made by Do-Rego and co-workers using dopamine uptake data. It appears that the isomerization phenomenon previously described for PE2I is not limited to tropane-based DAT inhibitors, but is, in fact, a general property of dopamine transporter protein, similar to "isomerization" process reported previously for G-protein coupled receptors. The rapid first step of association of the GBR 12909 is characterized by the equilibrium constant K(L)=34+/-11nM and the second slow step by k(i)=0.033+/-0.005s(-1).

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.

Ultrasonic evidence of hydrophobic interactions. Effect of ultrasound on benzoin condensation and some other reactions in aqueous ethanol.

The kinetics of KCN-catalyzed benzoin condensation of benzaldehyde in water and ethanol-water binary mixtures was investigated both under ultrasound at 22 kHz and without sonication. Thermodynamic activation parameters were calculated from kinetic data obtained at 35, 50, and 65 degrees C. Evidence that ultrasound can retard reactions is reported and hence a direct proof that sonochemical processes occur in the bulk solution. Former results and literature data for ester hydrolyses and tert-butyl chloride solvolysis are involved in the discussion. A quantitative relationship between sonication effects and the hydrophobicity of reagents is presented for the first time. Ultrasound affects hydrophobic interactions with the solvent, which are not manifested in conventional kinetics. When it suppresses the stabilization of the encounter complexes between reagents, sonication hinders the reaction but accelerates it when it perturbs the hydrophobic stabilization of the ground state of a reagent.

Synthesis of 3H-labeled N-(3-iodoprop-2E-enyl)-2beta-carbomethoxy-3beta-(4-methylphenyl)nortropane (PE2I) and its interaction with mice striatal membrane fragments.

PE2I is a high-affinity dopamine transporter (DA(T)) ligand that exhibits high selectivity for the DA(T) over the serotonin (5-HT(T)) and norephinephrine transporter (NE(T)) making its carbon-11 and iodine-125 labeled forms very useful for quantification of DA(T) binding sites in vivo. In this paper, we reported the synthesis of tritium-labeled PE2I, as well as improvements in the preparation of its acid precursor and PE2I itself with significant improvement in the total yield compared to previously published results. The new radioligand was evaluated as a tool for assessing DA(T) binding sites in vitro. The radioligand binding to mice striatal membranes demonstrated a homogeneous population of DA(T) binding sites with the K(d) value 9 nM.

Comparison of cAMP-dependent protein kinase substrate specificity in reaction with proteins and synthetic peptides.

Mutants of L-type pyruvate kinase with modified peptide sequence around the Ser-12 phosphorylation site were prepared and kinetics of their phosphorylation by protein kinase A was studied. The profile of substrate specificity obtained for these proteins was compared with the kinetic data of phosphorylation of short peptide substrates. Alterations made in protein structure caused weaker effects than the corresponding alterations made in peptides, while the amino acid preferences and the overall specificity pattern remained similar in the both cases. Thus, similar consensus motif holds for both protein and peptide substrates, but is less critical for recognition of proteins if compared with short peptides.

Slow isomerization step in the interaction between mouse dopamine transporter and dopamine re-uptake inhibitor N-(3-iodoprop-2E-enyl)-2beta-carbo-[3H]methoxy-3beta-(4'-methylphenyl)nortropane.

The kinetics of the association and dissociation of the tritium-labeled selective and potent dopamine transporter inhibitor N-(3-iodoprop-2E-enyl)-2beta-carbo-[3H]methoxy-3beta-(4'-methylphenyl)nortropane ([3H]PE2I) with the transporter of mouse striatal membranes was studied. The analysis revealed that the specific binding of [3H]PE2I occurs within a homogeneous population of binding sites in these membranes. The relatively slow binding process was characterized by the pseudo-first-order rate constant kobs. The plot of these rate constants versus free radioligand concentration was hyperbolic, demonstrating that at least two kinetically distinguishable steps can be identified in the interaction of dopamine transporter with this inhibitor. The fast and reversible binding step, characterized by dissociation constant KA = 51 +/- 23 nM, is followed by a slow but also reversible isomerization step of the complex, characterized by the isomerization rate constant ki = (7 +/- 2)10(-2) s(-1) and by the rate constant k(-i) = (3.9 +/- 0.5)10(-3) s(-1) for the reverse process. This isomerization step increases the apparent affinity of the ligand and probably consists of a conformational transition of the transporter protein, induced by the inhibitor molecule.

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.

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.

Screening for the optimal specificity profile of protein kinase C using electrospray mass-spectrometry.

A peptide library approach based on electrospray mass-spectrometric (ESI-MS) detection of phosphopeptides was designed for rapid and quantitative characterization of protein kinase specificity. The k(cat)/K(m) values for the protein kinase Cbeta (PKCbeta) were determined for a systematically varied set of individual substrate peptides in library mixtures by the ESI-MS method. The analysis revealed a complex structural specificity profile in positions around the phosphorylated serine with hydrophobic and/or basic residues being mostly preferred. On the basis of the kinetic parameters, a highly efficient peptide substrate for PKCbeta (K(m)value below 100 nM) FRRRRSFRRR and its alanine substituted pseudosubstrate-analog inhibitor (K(i) value of 76 nM) were designed. The quantitative specificity profiles obtained by the new approach contained more information about kinase specificity than the conventional substrate consensus motifs. The new method presents a promising basis for design of substrate-site directed peptide or peptidomimetic inhibitors of protein kinases. Second, highly specific substrates could be designed for novel applications such as high-throughput protein kinase activity screens on protein kinase chips.

Similar dynamics of G-protein coupled receptors molecules in response to antagonist binding.

In this communication we would like to draw attention on surprisingly similar transition of G-protein coupled receptors (GPCR) molecules, observed in these kinetic studies made with different receptors and their antagonists.

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.

Kinetic analysis of [35S]dATP alpha S interaction with P2y(1) nucleotide receptor.

The kinetics of 2'-deoxyadenosine-5'-O-(1-thiotriphosphate) ([(35)S]dATP alpha S) interaction with membrane fragments of transfected astrocytoma 1321N1 cells, expressing human P2Y(1) receptors, and the same wild-type cells, not expressing P2Y receptors were studied. Binding of this radioligand was observed with both types of membranes, but sites showing slow on-rate were found only on the transfected cells. These "slow" binding sites behaved as a kinetically homogeneous population and their interaction with the radioligand was shown to occur in two steps, R+A(K(A))<==>RA(k(i))<==>(k(-i))(RA), including the relatively slow isomerization of the complex RA into (RA). Evidence was obtained to assign the isomerized ("slow") binding sites on the transfected cells as P2Y(1) receptor sites, differentiated from other binding sites of non-receptor origin by kinetic analysis, and characterised by the kinetic parameters K(A)=59 +/- 19 nM, k(i)=(9.0 +/- 0.8)10(-3)s(-1) and k(-i)=(3.9 +/- 0.7)10(-3)s(-1). [(35)S]dATP alpha S binding, with kinetic criteria, can be of value for differentiation of the receptor sites from non-receptor sites and thus provides solid basis for radioligand assay of P2Y(1) receptors.