Halogen bonds involved in binding of halogenated ligands by protein kinases.

Analysis of 664 known structures of protein kinase complexes with halogenated ligands revealed 424 short contacts between a halogen atom and a potential protein X-bond acceptor, the topology and geometry of which were analyzed according to the type of a halogen atom (X = Cl, Br, I) and a putative protein X-bond acceptor. Among 236 identified halogen bonds, the most represented ones are directed to backbone carbonyls of the hinge region and may replace the pattern of ATP-like hydrogen bonds. Some halogen-π interactions with either aromatic residues or peptide bonds, that accompany the interaction with the hinge region, may possibly enhance ligand selectivity. Interestingly, many of these halogen-π interactions are bifurcated. Geometrical preferences identify iodine as the strongest X-bond donor, less so bromine, while virtually no such preferences were observed for chlorine; and a backbone carbonyl as the strongest X-bond acceptor. The presence of a halogen atom in a ligand additionally affects the properties of proximal hydrogen bonds, which according to geometrical parameters get strengthened, when a nitrogen of a halogenated ligand acts as the hydrogen bond donor.

UV-Vis spectroscopy of tyrosine side-groups in studies of protein structure. Part 2: selected applications.

In Part 2 we discuss application of several different types of UV-Vis spectroscopy, such as normal, difference, and second-derivative UV absorption spectroscopy, fluorescence spectroscopy, linear and circular dichroism spectroscopy, and Raman spectroscopy, of the side-chain of tyrosine residues in different molecular environments. We review the ways these spectroscopies can be used to probe complex protein structures.

UV-Vis spectroscopy of tyrosine side-groups in studies of protein structure. Part 1: basic principles and properties of tyrosine chromophore.

Spectroscopic properties of tyrosine residues may be employed in structural studies of proteins. Here we discuss several different types of UV-Vis spectroscopy, like normal, difference and second-derivative UV absorption spectroscopy, fluorescence spectroscopy, linear and circular dichroism spectroscopy, and Raman spectroscopy, and corresponding optical properties of the tyrosine chromophore, phenol, which are used to study protein structure.

Properties of phosphorylated thymidylate synthase.

Thymidylate synthase (TS) may undergo phosphorylation endogenously in mammalian cells, and as a recombinant protein expressed in bacterial cells, as indicated by the reaction of purified enzyme protein with Pro-Q® Diamond Phosphoprotein Gel Stain (PGS). With recombinant human, mouse, rat, Trichinella spiralis and Caenorhabditis elegans TSs, expressed in Escherichia coli, the phosphorylated, compared to non-phosphorylated recombinant enzyme forms, showed a decrease in Vmax(app), bound their cognate mRNA (only rat enzyme studied), and repressed translation of their own and several heterologous mRNAs (human, rat and mouse enzymes studied). However, attempts to determine the modification site(s), whether endogenously expressed in mammalian cells, or recombinant proteins, did not lead to unequivocal results. Comparative ESI-MS/analysis of IEF fractions of TS preparations from parental and FdUrd-resistant mouse leukemia L1210 cells, differing in sensitivity to inactivation by FdUMP, demonstrated phosphorylation of Ser(10) and Ser(16) in the resistant enzyme only, although PGS staining pointed to the modification of both L1210 TS proteins. The TS proteins phosphorylated in bacterial cells were shown by (31)P NMR to be modified only on histidine residues, like potassium phosphoramidate (KPA)-phosphorylated TS proteins. NanoLC-MS/MS, enabling the use of CID and ETD peptide fragmentation methods, identified several phosphohistidine residues, but certain phosphoserine and phosphothreonine residues were also implicated. Molecular dynamics studies, based on the mouse TS crystal structure, allowed one to assess potential of several phosphorylated histidine residues to affect catalytic activity, the effect being phosphorylation site dependent.

Thermodynamics parameters for binding of halogenated benzotriazole inhibitors of human protein kinase CK2α.

The interaction of human CK2α (hCK2α) with nine halogenated benzotriazoles, TBBt and its analogues representing all possible patterns of halogenation on the benzene ring of benzotriazole, was studied by biophysical methods. Thermal stability of protein-ligand complexes, monitored by calorimetric (DSC) and optical (DSF) methods, showed that the increase in the mid-point temperature for unfolding of protein-ligand complexes (i.e. potency of ligand binding to hCK2α) follow the inhibitory activities determined by biochemical assays. The dissociation constant for the ATP-hCK2α complex was estimated with the aid of microscale thermophoresis (MST) as 4.3±1.8 µM, and MST-derived dissociation constants determined for halogenated benzotriazoles, when converted according to known ATP concentrations, perfectly reconstruct IC50 values determined by the biochemical assays. Ligand-dependent quenching of tyrosine fluorescence, together with molecular modeling and DSC-derived heats of unfolding, support the hypothesis that halogenated benzotriazoles bind in at least two alternative orientations, and those that are efficient hCK2α inhibitors bind in the orientation which TBBt adopts in its complex with maize CK2α. DSC-derived apparent heat for ligand binding (ΔΔHbind) is driven by intermolecular electrostatic interactions between Lys68 and the triazole ring of the ligand, as indicated by a good correlation between ΔΔHbind and ligand pKa. Overall results, additionally supported by molecular modeling, confirm that a balance of hydrophobic and electrostatic interactions contribute predominantly (~40 kJ/mol), relative to possible intermolecular halogen/hydrogen bonding (less than 10 kJ/mol), in binding of halogenated benzotriazoles to the ATP-binding site of hCK2α. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases.

Thermodynamic parameters for binding of some halogenated inhibitors of human protein kinase CK2.

The interaction of human CK2α with a series of tetrabromobenzotriazole (TBBt) and tetrabromobenzimidazole (TBBz) analogs, in which one of the bromine atoms proximal to the triazole/imidazole ring is replaced by a methyl group, was studied by biochemical (IC50) and biophysical methods (thermal stability of protein-ligand complex monitored by DSC and fluorescence). Two newly synthesized tri-bromo derivatives display inhibitory activity comparable to that of the reference compounds, TBBt and TBBz, respectively. DSC analysis of the stability of protein-ligand complexes shows that the heat of ligand binding (Hbind) is driven by intermolecular electrostatic interactions involving the triazole/imidazole ring, as indicated by a strong correlation between Hbind and ligand pKa. Screening, based on fluorescence-monitored thermal unfolding of protein-ligand complexes, gave comparable results, clearly identifying ligands that most strongly bind to the protein. Overall results, additionally supported by molecular modeling, confirm that a balance of hydrophobic and electrostatic interactions contribute predominantly, relative to possible intermolecular halogen bonding, in binding of the ligands to the CK2α ATP-binding site.

8-Azapurines as isosteric purine fluorescent probes for nucleic acid and enzymatic research.

The 8-azapurines, and their 7-deaza and 9-deaza congeners, represent a unique class of isosteric (isomorphic) analogues of the natural purines, frequently capable of substituting for the latter in many biochemical processes. Particularly interesting is their propensity to exhibit pH-dependent room-temperature fluorescence in aqueous medium, and in non-polar media. We herein review the physico-chemical properties of this class of compounds, with particular emphasis on the fluorescence emission properties of their neutral and/or ionic species, which has led to their widespread use as fluorescent probes in enzymology, including enzymes involved in purine metabolism, agonists/antagonists of adenosine receptors, mechanisms of catalytic RNAs, RNA editing, etc. They are also exceptionally useful fluorescent probes for analytical and clinical applications in crude cell homogenates.

A Protein Data Bank survey reveals shortening of intermolecular hydrogen bonds in ligand-protein complexes when a halogenated ligand is an H-bond donor.

Halogen bonding in ligand-protein complexes is currently widely exploited, e.g. in drug design or supramolecular chemistry. But little attention has been directed to other effects that may result from replacement of a hydrogen by a strongly electronegative halogen. Analysis of almost 30000 hydrogen bonds between protein and ligand demonstrates that the length of a hydrogen bond depends on the type of donor-acceptor pair. Interestingly, lengths of hydrogen bonds between a protein and a halogenated ligand are visibly shorter than those estimated for the same family of proteins in complexes with non-halogenated ligands. Taking into account the effect of halogenation on hydrogen bonding is thus important when evaluating structural and/or energetic parameters of ligand-protein complexes. All these observations are consistent with the concept that halogenation increases the acidity of the proximal amino/imino/hydroxyl groups and thus makes them better, i.e. stronger, H-bond donors.

Myristoylated derivatives of 2',3'-didehydro-2',3'-dideoxythymidine (stavudine) bi-functional prodrugs with potent anti-HIV-1 activity and low cytotoxicity.

BACKGROUND: To improve in vitro antiviral activity and selectivity of stavudine (d4T), a range of its bi-functional prodrugs, 5'-O-myristoylated derivatives, have been synthesized. METHODS: Stavudine 5'-O-myristoylated esters were synthesized using modified Parang's procedure. The cytotoxicity and anti-HIV activity was evaluated in the established MT-4 cell line. The level of p24 protein in culture medium was assayed, and EC50 and EC90 values were determined. RESULTS: Excellent anti-HIV activity was obtained for stavudine derivatives 2',3'-didehydro-2',3'-dideoxy-5'-O-(11-thioethylundecanoyl) thymidine, 2',3'-didehydro-2',3'-dideoxy-5'-O-(12-thioethyldodecanoyl) thymidine and 5'-O-(12-azidododecanoyl)-2',3'-didehydro-2',3'-dideoxythymidine with C10 and C11 alkyl chains bearing thioethyl- and azido- substituents. These prodrugs were more potent than the parent stavudine, as is clear from their EC50 values: 2',3'-didehydro-2',3'-dideoxy-5'-O-(11-thioethylundecanoyl) thymidine (R=CO(CH2)10SC2H5, EC50 0.06 µM), 2',3'-didehydro-2',3'-dideoxy-5'-O-(12-thioethyldodecanoyl) thymidine (R=CO(CH2)11SC2H5, EC50 0.09 µM) and 5'-O-(12-azidododecanoyl)-2',3'-didehydro-2',3'-dideoxythymidine (R=CO(CH2)11N3, EC50 0.06 µM), while 50% cytotoxic concentration was >16.65 µM, >7.5 µM and >18.53 µM, respectively. CONCLUSIONS: Overall data demonstrate that compounds 2',3'-didehydro-2',3'-dideoxy-5'-O-(11-thioethylundecanoyl) thymidine, 2',3'-didehydro-2',3'-dideoxy-5'-O-(12-thioethyldodecanoyl) thymidine and 5'-O-(12-azidododecanoyl)-2',3'-didehydro-2',3'-dideoxythymidine are very potent and selective anti-HIV agents and could be useful in treatment of HIV infections of the central nervous system.

Halogen bonding at the ATP binding site of protein kinases: preferred geometry and topology of ligand binding.

Halogenated ligands have been widely developed as potent, and frequently selective, inhibitors of protein kinases (PK). Herein, all structures of protein kinases complexed with a halogenated ligand, identified in the PDB, were analyzed in the context of eventual contribution of halogen bonding to protein-ligand interactions. Global inspection shows that two carbonyl groups of residues located in the hinge region are the most abundant halogen bond acceptors. In contrast to solution data, well-defined water molecules, located at sites conserved across most PK structures, are also involved in halogen bonding. Analysis of cumulative distributions of halogen-acceptor distances shows that structures displaying short contacts involving a halogen atom are overpopulated, contributing together to clearly defined maxima of 2.82, 2.91 and 2.94Å for chlorine, bromine and iodine, respectively. The angular preference of a halogen bond favors ideal topology (180°, 120°) for iodine. For bromine the distribution is much more dispersed, and no such preference was found for chlorine. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).

Resolving differences in substrate specificities between human and parasite phosphoribosyltransferases via analysis of functional groups of substrates and receptors.

We herein review experimental and theoretical approaches widely applied to delineation of the differences in substrate specificities between human and parasite phosphoribosyltransferases (PRTases), the latter of which are key targets for treatment of diseases caused by parasites. Standard Molecular Dynamics (MD) simulations have been applied to determine why the human PRTase prefers guanine over xanthine, whereas the Tritrichomonas foetus enzyme exhibits only a slight preference. We analyze this problem with the aid of standard MD simulations, as well as constant-pH MD simulations. Comparison of results of the two approaches reveals substantial differences, e.g. several Asp and Glu residues in the parasite enzyme, and one Glu residue in the human enzyme, are predicted to be permanently or frequently protonated during constant-pH simulations, whereas standard MD simulations assume that these residues are always ionized. Most interesting is the observation of a large conformational change, leading to tighter binding of the ligand, observed in constant-pH MD simulations of the parasite PRTase complexed with XMP, and lack of such a change in the human enzyme complexed with XMP.

Isomeric mono-, di-, and tri-bromobenzo-1H-triazoles as inhibitors of human protein kinase CK2α.

To further clarify the role of the individual bromine atoms of 4,5,6,7-tetrabromotriazole (TBBt), a relatively selective inhibitor of protein kinase CK2, we have examined the inhibition (IC(50)) of human CK2α by the two mono-, the four di-, and the two tri- bromobenzotriazoles relative to that of TBBt. Halogenation of the central vicinal C(5)/C(6) atoms proved to be a key factor in enhancing inhibitory activity, in that 5,6-di-Br(2)Bt and 4,5,6-Br(3)Bt were almost as effective inhibitors as TBBt, notwithstanding their marked differences in pK(a) for dissociation of the triazole proton. The decrease in pK(a) on halogenation of the peripheral C(4)/C(7) atoms virtually nullifies the gain due to hydrophobic interactions, and does not lead to a decrease in IC(50). Molecular modeling of structures of complexes of the ligands with the enzyme, as well as QSAR analysis, pointed to a balance of hydrophobic and electrostatic interactions as a discriminator of inhibitory activity. The role of halogen bonding remains debatable, as originally noted for the crystal structure of TBBt with CK2α (pdb1j91). Finally we direct attention to the promising applicability of our series of well-defined halogenated benzotriazoles to studies on inhibition of kinases other than CK2.

Synthesis and physico-chemical properties in aqueous medium of all possible isomeric bromo analogues of benzo-1H-triazole, potential inhibitors of protein kinases.

In ongoing studies on the role of the individual bromine atoms of 4,5,6,7-tetrabromobenzotriazole (TBBt) in its relatively selective inhibition of protein kinase CK2α, we have prepared all the possible two mono-, four di-, and two tri-bromobenzotriazoles and determined their physicochemical properties in aqueous medium. They exhibited a general trend of a decrease in solubility with an increase in the number of bromines on the benzene ring, significantly modulated by the pattern of substitution. For a given number of attached bromines, this was directly related to the electronic effects resulting from different sites of substitution, leading to marked variations of pK(a) values for dissociation of the triazole proton. Experimental data (pK(a), solubility) and ab initio calculations demonstrated that hydration of halogenated benzotriazoles is driven by a subtle balance of hydrophobic and polar interactions. The combination of QM-derived free energies for solvation and proton dissociations was found to be a reasonably good predictor of inhibitory activity of halogenated benzotriazoles vs CK2α. Since the pattern of halogenation of the benzene ring of benzotriazole has also been shown to be one of the determinants of inhibitory potency vs some viruses and viral enzymes, the present comprehensive description of their physicochemical properties should prove helpful in efforts to elucidate reaction mechanisms, including possible halogen bonding, and the search for more selective and potent inhibitors.

Poisson-Boltzmann continuum-solvation models: applications to pH-dependent properties of biomolecules.

All molecules can be viewed as either discrete or continuous assemblies of electric charges, and electrostatics plays a major role in intermolecular and intramolecular interactions. Moreover, charge distribution within molecules may fluctuate due to the presence of ionizable groups capable of exchanging protons with the environment, leading to pH-dependence of phenomena involving such molecules. Electrostatic aspects of complex shapes and environments of biological molecules, in vitro and in vivo, are relatively well amenable to treatment by Poisson-Boltzmann models, which are attractive in that they possess a clear physical meaning, and can be readily solved by several mathematically sound methods. Here we describe applications of these models to obtain valuable insights into some biologically important pH-dependent properties of biomolecules, such as stability, binding of ligands (including potential drugs), enzymatic activity, conformational transitions, membrane transport and viral entry.

Relative role of halogen bonds and hydrophobic interactions in inhibition of human protein kinase CK2α by tetrabromobenzotriazole and some C5-substituted analogues.

To examine the relative role of halogen bonding and hydrophobic interactions in the inhibition of human CK2alpha by 4,5,6,7-tetrabromobenzotriazole (TBBt), we have synthesized a series of 5-substituted benzotriazoles (Bt) and the corresponding 5-substituted 4,6,7-tribromobenzotriazoles (Br3Bt) and examined their inhibition of human CK2alpha relative to that of TBBt. The various C(5) substituents differ in size (H and CH3), electronegativity (NH2 and NO2), and hydrophobicity (COOH and Cl). Some substituents were halogen bond donors (Cl, Br), while others were fluorine bond donors (F and CF3). Most of the 5-substituted analogues of Br3Bt (with the exception of COOH and NH2) exhibited inhibitory activity comparable to that of TBBt, whereas the 5-substituted analogues of the parent Bt were only weakly active (Br, Cl, NO2, CF3) or inactive. The observed effect of the volume of a ligand molecule pointed to its predominant role in inhibitory activity, indicating that presumed halogen bonding, identified in crystal structures and by molecular modeling, is dominated by hydrophobic interactions. Extended QSAR analysis additionally pointed to the monoanion and a preference for the N(1)-H protomer of the neutral ligand as parameters crucial for prediction of inhibitory activity. This suggests that the monoanions of TBBt and its congeners are the active forms that efficiently bind to CK2alpha, and the binding affinity is coupled with protomeric equilibrium of the neutral ligand.

Phosphorylation of thymidylate synthase from various sources by human protein kinase CK2 and its catalytic subunits.

Thymidylate synthase (TS) was found to be a substrate for both catalytic subunits of human CK2, with phosphorylation by CK2alpha and CK2alpha' characterized by similar K(m) values, 4.6microM and 4.2microM, respectively, but different efficiencies, the apparent turnover number with CK2alpha being 10-fold higher. With both catalytic subunits, phosphorylation of human TS, like calmodulin and BID, was strongly inhibited in the presence of the regulatory subunit CK2beta, the holoenzyme being activated by polylysine. Phosphorylation of recombinant human, rat, mouse and Trichinella spiralis TSs proteins was compared, with the human enzyme being apparently a much better substrate than the others. Following hydrolysis and TLC, phosphoserine was detected in human and rat, and phosphotyrosine in T. spiralis, TS, used as substrates for CK2alpha. MALDI-TOF MS analysis led to identification of phosphorylated Ser(124) in human TS, within a sequence LGFS(124)TREEGD, atypical for a CK2 substrate recognition site. The phosphorylation site is located in a region considered important for the catalytic mechanism or regulation of human TS, corresponding to the loop 107-128. Following phosphorylation by CK2alpha, resulting in incorporation of 0.4mol of phosphate per mol of dimeric TS, human TS exhibits unaltered K(m) values for dUMP and N(5,10)-methylenetetrahydrofolate, but a 50% lower turnover number, pointing to a strong influence of Ser(124) phosphorylation on its catalytic efficiency.

Fluorescence emission properties of 8-aza analogues of caffeine, theophylline, and related N-alkyl xanthines.

Fluorescence emission properties of 8-azacaffeine, 8-azatheophylline and other N-alkylated 8-azaxanthines (8-azaXan) have been examined. It is shown that N-methylated 8-azaxanthines, as well as 8-azatheophylline, are highly fluorescent in aqueous medium as the neutral, and, in some instances, also as the monoanionic, forms. 8-Azacaffeine exhibits moderate emission, but its isomer, 1,3,8-trimethyl-8-azaXan, is highly fluorescent. All three 8-azaxanthines monomethylated on the triazole ring, as well as 8-azaxanthosine, exhibit increased acidity in the excited state. Some fluorescent pyrazolo[4,3-d]pyrimidine-5,7-diones, xanthine congeners of pyrazolo[4,3-d]pyrimidines, are also reported. Many of these are good fluorescent probes in enzymatic, receptor binding, and nucleic acid systems, some examples of which are presented. In particular, 8-azaXan is an excellent fluorescent probe for purine nucleoside phosphorylases, as a fluorogenic substrate in the reverse, synthetic pathway.