Polyphosphates and pyrophosphates of pentopyranoses and pentofuranoses as allosteric effectors of human hemoglobin: synthesis, molecular recognition, and oxygen release

Perphosphorylated pentopyranoses and pentofuranoses were synthesized from parent carbohydrates as potential allosteric effectors of hemoglobin (Hb). The construction of seven- and eight-membered cyclic pyrophosphates was also carried out successfully on most of the pentoses. All final compounds were tested for their efficiency on oxygen release from human Hb. Most proved to be efficient allosteric effectors, some of them with an affinity toward Hb and an effect on oxygen release from Hb approaching that of myo-inositol hexakisphosphate, which is one of the most active allosteric effectors of Hb. The efficacy was higher for free phosphates than for pyrophosphates.     

Tetrakisphosphates and bispyrophosphates of myo-inositol derivatives as allosteric effectors of human hemoglobin: Synthesis, molecular recognition, and oxygen release

Various 2,5- and 1,4-substituted and unsubstituted myo-inositol tetrakisphosphates and bispyrophosphates were prepared following a general synthetic pathway. All final compounds were tested for their capability to induce oxygen release from human hemoglobin. Most of these proved to be efficient allosteric effectors, with similar affinities for hemoglobin to that of myo-inositol hexakisphosphate, which is one of the best known allosteric effectors of hemoglobin. The efficacy was found to be higher for free phosphates than pyrophosphates. As allosteric Hb effectors, these compounds enable enhanced oxygen release. These effects increase with the strength of Hb binding and correspond primarily to electrostatic interactions. Stereochemical and steric factors also play a significant but secondary role in molecular recognition. In view of the central role played by hypoxia in numerous types of diseases, the exploration of myo-inositol phosphate derivatives represents an important avenue in the search for substances which act on the oxygenation status of tissues and may have significant potential in the discovery and development of novel drug candidates.     

Covalent Allosteric Inhibitors of Akt Generated Using a Click Fragment Approach

Akt is a protein kinase that has been implicated in the progression of cancerous tumours. A number of covalent allosteric Akt inhibitors are known, and based on these scaffolds, a small library of novel potential covalent allosteric imidazopyridine-based inhibitors was designed. The envisaged compounds were synthesised, with click chemistry enabling a modular approach to a number of the target compounds. The binding modes, potencies and antiproliferative activities of these synthesised compounds were explored, thereby furthering the structure activity relationship knowledge of this class of Akt inhibitors. Three novel covalent inhibitors were identified, exhibiting moderate activity against Akt1 and various cancer cell lines, potentially paving the way for future covalent allosteric inhibitors with improved properties.     

Central nicotinic receptors: structure, function, ligands, and therapeutic potential

The growing interest in nicotinic receptors, because of their wide expression in neuronal and non-neuronal tissues and their involvement in several important CNS pathologies, has stimulated the synthesis of a high number of ligands able to modulate their function. These membrane proteins appear to be highly heterogeneous, and still only incomplete information is available on their structure, subunit composition, and stoichiometry. This is due to the lack of selective ligands to study the role of nAChR under physiological or pathological conditions; so far, only compounds showing selectivity between alpha4beta2 and alpha7 receptors have been obtained. The nicotinic receptor ligands have been designed starting from lead compounds from natural sources such as nicotine, cytisine, or epibatidine, and, more recently, through the high-throughput screening of chemical libraries. This review focuses on the structure of the new agonists, antagonists, and allosteric ligands of nicotinic receptors, it highlights the current knowledge on the binding site models as a molecular modeling approach to design new compounds, and it discusses the nAChR modulators which have entered clinical trials.     

Sodium dialkyl phosphates: Surfactant properties and use in heavy duty detergents

A study of the physical and surface active properties of the sodium salts of dialkyl phosphates has been made. These compounds have been evaluated for use as surfactants in built detergents and their performance has been compared with commercial surfactants. The dialkyl hydrogen phosphates contained from 16–26 carbon atoms per molecule and were prepared from both straight chain alcohols and commercially available branched chain alcohols. In general, the surface active properties of the branched chain compounds are better than their corresponding straight chain compounds. Good detergency performance is obtained when the number of carbon atoms per molecule is between 17 and 23. Optimum performance is obtained with the symmetrical didecyl hydrogen phosphate salts made from commercial decyl alcohol produced by the oxo-process.     

A Novel Approach to the Discovery of Small-Molecule Ligands of CDK2

In an attempt to identify novel small-molecule ligands of cyclin-dependent kinase 2 (CDK2) with potential as allosteric inhibitors, we have devised a robust and cost-effective fluorescence-based high-throughput screening assay. The assay is based on the specific interaction of CDK2 with the extrinsic fluorophore 8-anilino-1-naphthalene sulfonate (ANS), which binds to a large allosteric pocket adjacent to the ATP site. Hit compounds that displace ANS directly or indirectly from CDK2 are readily classified as ATP site binders or allosteric ligands through the use of staurosporine, which blocks the ATP site without displacing ANS. Pilot screening of 1453 compounds led to the discovery of 12 compounds with displacement activities (EC(50) values) ranging from 6 to 44 μM, all of which were classified as ATP-site-directed ligands. Four new type I inhibitor scaffolds were confirmed by X-ray crystallography. Although this small compound library contained only ATP-site-directed ligands, the application of this assay to large compound libraries has the potential to reveal previously unrecognized chemical scaffolds suitable for structure-based design of CDK2 inhibitors with new mechanisms of action.     

Flame retardancy effects of halogenated phosphates on poly(ethylene terephthalate) fabric

A Chemical research on the flame retardancy effect of halogenated phosphates on poly(ethylene terephathalate) fabric was carried out by thermogravimetry, infrared spectral analysis, and mass spectrometry. The following conclusions were drawn: (1) The flame retardancy effect of halogenated phosphates such as tris(2,3-dibromopropyl) phosphate is due to altering the pyrolysis reaction of polyester by aldol condensation. (2) The probability that liberated halogen compounds from the phosphates act as radical acceptors in a flame zone may be low. (3) Incorporation of halogen elements into phosphates appear to depress evaporation of phosphates, which act as acidic catalysts in aldol condensation from the condensed phase.     

The initial and residual agronomic effectiveness of some Indian,USA and Australian rock phosphates

The initial and residual agronomic effectiveness of six apatite rock phosphates from India, one from the USA (North Carolina) and one from Australia (Queensland) were evaluated in a pot trial with wheat on a lateritic soil. All of the Indian rock phosphates were very poor sources of phosphorus. Values of initial effectiveness relative to monocalcium phosphate ranged from < 0.0001 to 0.02 and from < 0.0001 to 0.008 for measurements based on yield and phosphorus uptake respectively. The residual effectiveness relative to freshly applied monocalcium phosphate was determined by growing a second crop on the fertilized soils. The effectiveness of the Indian rock phosphates remained very low ranging from < 0.0001 to 0.002 and from < 0.0001 to 0.0004 for yield and phosphorus uptake respectively. Queensland and North Carolina rock phosphates were much superior to the Indian sources with initial effectiveness values in terms of yield of 0.08 and 0.37 and residual effectiveness values of 0.02 and 0.15 respectively. For each crop there was a single relationship between yield and phosphorus uptake (i.e. internal efficiency) for all phosphorus sources showing that variations in yield response were due solely to differences in phosphorus availability. Sodium bicarbonate extractable phosphorus values for fertilized soils sampled shortly after fertilization were not predictive of yield unless different calibration curves were used for the different phosphorus fertilizers.     

Engineering Allosteric Regulation into Biological Catalysts

Enzymes and ribozymes constitute two classes of biological catalysts. The activity of many natural enzymes is regulated by the binding of ligands that have different structures than their substrates; these ligands are consequently called allosteric effectors. In most allosteric enzymes, the allosteric binding site lies far away from the active site. This implies that communication pathways must exist between these sites. While mechanisms of allosteric regulation were developed more than forty years ago, they continue to be revisited regularly. The improved understanding of these mechanisms has led in the past two decades to projects to transform several unregulated enzymes into allosterically regulated ones either by rational design or directed evolution techniques. More recently, ribozymes have also been the object of similar successful engineering efforts. In this review, after briefly summarising recent progress in the theories of allosteric regulation, several strategies to engineer allosteric regulations in enzymes and ribozymes are described and compared. These redesigned biological catalysts find applications in a variety of areas.     

Artificial Allosteric System 5. Allosteric Gain,Total Cooperativity and Local Cooperativity as Appropriate Indices for Allosteric Effect

Magnitude of cooperativity of a hypothetical m-subunit allosteric system of hemoglobin type is discussed, based on computation of the amount of O₂ to be transferred to the corresponding monomeric nonallosteric system of myoglobin type. For quantitative discussion, “allosteric gain” is defined as the difference between the amount of O₂ to be transferred from the allosteric to the nonallosteric system and that from the hypothetical system of average O₂ affinity to the nonallosteric system. These two quantities, allosteric gain and amount of O₂ transfer, are quite sensitive to equilibrium constant ratio, especially K_m/K₁, (K_i+1/K_i)_max, and i. Hill's coefficient appropriately describes neither the amount of O₂ transferred nor allosteric gain. Allosteric gain or the amount of O₂ transfer which is best characterized by the total cooperativity index (K_m/K₁) and the local cooperativity indices (K_i+1/K_i) seem to be appropriate measures of the magnitude of cooperativity.     

Novel Positive Allosteric Modulators of µ Opioid Receptor—Insight from In Silico and In Vivo Studies

Opioids are the drugs of choice in severe pain management. Unfortunately, their use involves serious, potentially lethal side effects. Therefore, efforts in opioid drug design turn toward safer and more effective mechanisms, including allosteric modulation. In this study, molecular dynamics simulations in silico and ‘writhing’ tests in vivo were used to characterize potential allosteric mechanism of two previously reported compounds. The results suggest that investigated compounds bind to μ opioid receptor in an allosteric site, augmenting action of morphine at subeffective doses, and exerting antinociceptive effect alone at higher doses. Detailed analysis of in silico calculations suggests that first of the compounds behaves more like allosteric agonist, while the second compound acts mainly as a positive allosteric modulator.     

Herbivorous Caterpillars and the Green Leaf Volatile (GLV) Quandary

Several herbivorous caterpillars contain effectors in their oral secretions that alter the emission of green leaf volatiles (GLVs) produced by the plants upon which the caterpillars are feeding. These effectors include an isomerase, a fatty acid dehydratase (FHD), and a heat-stable hexenal trapping (HALT) molecule. GLVs serve as signaling compounds in plant-insect interactions and inter-and intra-plant communication. However, it is not known whether these GLV-altering effectors are common among herbivorous caterpillars, or the evolutionary context of these effectors in relation to GLV emission by host plants in response to feeding damage. Here, we examined the distribution and activity of the isomerase, FHD, and HALT effectors across 10 species spanning 7 lepidopteran families. Six of the 10 species possessed all three effectors in their oral secretions. Activity from the HALT and FHD effectors was observed in all examined caterpillar species, while activity from the isomerase effector varied in some species and was absent in others. There was no discernable pattern in effector activity based on evolutionary divergence, since individual species within a family did not possess similar mechanisms to alter GLV emission. These data, demonstrating the GLV-altering effectors acting at different steps in the GLV biosynthetic pathway and present in the examined caterpillar species at different combinations with different activities, highlight the importance of these effectors in changing the emission of these compounds during caterpillar herbivory. Understanding the prevalence and roles of GLV-altering effectors and GLV emission itself will open new research areas in the dynamics of plant-insect interactions.

     

A novel mechanism of allosteric regulation of archaeal phosphoenolpyruvate carboxylase: a combined approach to structure-based alignment and model assessment

Phosphoenolpyruvate carboxylase (PEPC) catalyzes the irreversible carboxylation of phosphoenolpyruvate (PEP) and plays a crucial role in fixing atmospheric CO(2) in C(4) and CAM plants. The enzyme is widespread in plants and bacteria and mostly regulated allosterically by both positive and negative effectors. Archaeal PEPCs (A-PEPCs) have unique characteristics in allosteric regulation and molecular mass, distinct from their bacterial and eukaryote homologues, and their amino acid sequences have become available only recently. In this paper, we generated a structure-based alignment of archaeal, bacterial and eukaryote PEPCs and built comparative models using a combination of fold recognition, sequence and structural analysis tools. Our comparative modeling analysis identified A-PEPC-specific strong interactions between the two loops involved in both allostery and catalysis, which explained why A-PEPC is not influenced by any allosteric activators. We also found that the side-chain located three residues before the C-terminus appears to play a key role in determining the sensitivity to allosteric inhibitors. In addition to these unique features, we revealed how archaeal, bacterial and eukaryote PEPCs would share a common catalytic mechanism and adopt a similar mode of tetramer formation, despite their divergent sequences. Our novel observations will help design more efficient molecules for ecological and industrial use.     

Allosteric Regulation of Proteases

Allostery is a basic principle of control of enzymatic activities based on the interaction of a protein or small molecule at a site distinct from an enzyme's active center. Allosteric modulators represent an alternative approach to the design and synthesis of small‐molecule activators or inhibitors of proteases and are therefore of wide interest for medicinal chemistry. The structural bases of some proteinaceous and small‐molecule allosteric protease regulators have already been elucidated, indicating a general mechanism that might be exploitable for future rational design of small‐molecule effectors.     

Modulation of Ultrafast Conformational Dynamics in Allosteric Interaction of Gal Repressor Protein with Different Operator DNA Sequences

Although all forms of dynamical behaviour of a protein under allosteric interaction with effectors are predicted, little evidence of ultrafast dynamics in the interaction has been reported. Here, we demonstrate the efficacy of a combined approach involving picosecond‐resolved FRET and polarisation‐gated fluorescence for the exploration of ultrafast dynamics in the allosteric interaction of the Gal repressor (GalR) protein dimer with DNA operator sequences O_E and O_I. FRET from the single tryptophan residue to a covalently attached probe IAEDANS at a cysteine residue in the C‐terminal domain of GalR shows structural perturbation and conformational dynamics during allosteric interaction. Polarisation‐gated fluorescence spectroscopy of IAEDANS and another probe (FITC) covalently attached to the operator directly revealed the essential dynamics for cooperativity in the protein–protein interaction. The ultrafast resonance energy transfer from IAEDANS in the protein to FITC also revealed different dynamic flexibility in the allosteric interaction. An attempt was made to correlate the dynamic changes in the protein dimers with O_E and O_I with the consequent protein–protein interaction (tetramerisation) to form a DNA loop encompassing the promoter segment.     

Prostaglandin and acyl chain effects on glutamate dehydrogenase activity

Prostaglandins A₁ (PGA₁), A₂, B₁, B₂, E₁, E₂, F_1α, F_2α, and 19 esterified natural fatty acids were tested as effectors of beef liver glutamate dehydrogenase (L-glutamate: NAD(P)⁺, oxidoreductase [deaminating], EC 1.4.1.3). All prostaglandins tested are found to activate the enzyme initially, but only PGA₂>PGB₂≥PGA₁ cause a subsequent time-dependent loss (not inhibition) of NADH oxidation activity. Both PGA₁ and PGA₂ desensitize glutamate dehydrogenase to allosteric activation by ADP, whereas PGA₂ and PGB₂ desensitize to allosteric inactivation by GTP. Preincubation of enzyme with diethylstilbestrol prevents the initial activation by the PG. Of the methyl esters, only prostaglandin precursors inactivated the enzyme. Simultaneous desensitization to the ADP and GTP allosteric effects resulted. Multiple esterification to glycerol or phospholipids enhanced the action of linoleoyl and diminished the action of linolenoyl chains. Preincubation of the PGA with glutathione or cysteine prevents the inactivation; i.e., the sulfhydryl binding region of the prostaglandin must be free for enzyme to be inactivated. Sulfhydryl reagents also protect the enzyme from the effects of the unsaturated acyl chains, and pHMB mimics acyl protection against GTP allosteric inactivation. Where the lipid effector is active against sulfhydryl groups, the desensitizations to the ADP and GTP allosteric effectors are reciprocal. The initial activation, subsequent inactivation and desensitization to ADP and GTP are all characteristic of binding in the estrogen-specific effector site, suggesting this site as the target for PG and acyl action. In the PGA₂ activation, the effect is found to be amplified by the cooperativity of the enzyme at 1 PG molecule/6 molecules of GDH. We conclude from the action of the PG and structural analogs that the initial activation of glutamate dehydrogenase is caused by α,β-unsaturated monoketo cyclopental structures. GTP inhibition is blocked primarily by diketo structures which eventually inactivate the enzyme. ADP activation is blocked by sulfhydryl binding of the unsaturated cyclopental keto structure of the PG. Appearance of a 270 nm absorbance simultaneous to the acyl effects on the enzyme suggests that conjugated unsaturations are responsible for the precursor's qualitatively similar action to that of the PG.     

Preparation and surface- active properties of new amphipathic compounds with two phosphate groups and two long- chain alkyl groups

New compounds bearing two dihydrogen phosphate groups and two long- chain alkyl groups (octyl or decyl) were prepared in good yields by the reaction of 1,ω-bis(alkyloxymethyl)- oligo (ethylene glycol)s with polyphosphoric acid. Amphipathic di- or tetrasodium phosphates were obtained by neutralization of the free acids with sodium hydroxide, and their surface- active properties in water were measured. Almost all these di- or tetrasodium phosphates showed good water solubility. Their abilities to form micelles and to lower surface tension were fairly good as compared with general monoalkyl phosphates consisting of one longer alkyl chain and one hydrophilic group. The foaming property of aqueous solutions of the tetrasodium salts was different from that of the disodium salts. The former showed very low foaming ability.     

Communication Maps: Exploring Energy Transport through Proteins and Water

Frequency-resolved communication maps provide a coarse-grained, global mapping of energy transport channels in a protein as a function of frequency of modes that carry energy. We illustrate the approach with a study of the homodimeric hemoglobin of Scapharca inaequivalvis, which exhibits cooperativity during ligand binding. We compare energy transport between the two hemes of the unliganded and oxygenated protein, which is mediated by water as well as residues forming a hydrogen-bonding network at the interface between the globules, and lies along the pathway for allosteric transitions observed in time-resolved X-ray studies. Non-equilibrium molecular simulations on energy transport from the heme corroborate the energy transport pathways identified by the communication maps.     

Pyrolysis and combustion of cellulose. VII. Thermal analysis of the phosphorylation of cellulose and model carbohydrates during pyrolsis in the presence of aromatic phosphates and phosphoramides

Thermal gravimetric analysis, differential scanning calorimetry, and derivative thermal gravimetric analysis were utilized to characterize the thermal interactions between cellulose, 1-6, anhydro β-D-glucopyranoside, and D-glucose and model phosphate and phosphoramide flame retardants. The phosphoramides induced higher char yields than the phosphates during the pyrolysis of the mixtures of carbohydrates and organophosphorus compounds. Exothermic reactions attributed to phosphorylation and char formation were observed with each of the phosphoramide/carbohydrate mixtures and were absent with the phosphates. The individual phosphorus compounds studied showed similar thermal behavior with each of the carbohydrates indicating that the mode of interaction for these mixtures was similar. Isothermal gravimetric analysis of the organophosphorus/carbohydrate mixtures was used to measure the rate of decomposition weight loss from isothermal conditions. This weight loss was used as an indication of rate of fuel formation. The kinetics observed for these measurements indicated that the phosphoramide mixtures underwent a rapid weight loss to a final char with an effective E_act of about 55 kcal/mol while the phosphate mixtures exhibited effective E_act′s for decomposition lower than those observed for the pure carbohydrates. Mixtures of glucose with selcted arylphosphoramide esters were pyrolysed in order to determine the effect of lability of the leaving group on char formation. Gas chromatographic analysis of the pyrolysis products indicated that phenol was the favored leaving group in comparison with aniline units, but char promotion appeared to be dependent on the number of P-N bonds present in the original phosphoramide. Electron spectroscopy for chemical analysis indicated that chemically similar chars were obtained from the different organophosphorus/carbohydrate combinations.