Exploring QSAR with E-state index: selectivity requirements for COX-2 versus COX-1 binding of terphenyl methyl sulfones and sulfonamides

An attempt has been made to explore selectivity requirements for cyclooxygenase-2 (COX-2) versus cyclooxygenase-1 (COX-1) binding of terphenyl methyl sulfones and sulfonamides using electrotopological state (E-state) index and suitable indicator parameters. Multiple linear regression analyses produced statistically acceptable equations: the best relation based on 'all-possible-subsets regression' for COX-1 binding (n=18) showed predicted variance and explained variance of 0.675 and 0.777, respectively, while in case of the best equation for COX-2 binding (n=38), these values rose to 0.842 and 0.874, respectively. For the selectivity relation (n=17), predicted variance and explained variance values were 0.601 and 0.687, respectively. Based on the results of the analyses, three important sites have been suggested: sites A (methylsulfonyl or aminosulfonyl moiety), B (central phenyl ring), and C (terminal phenyl ring containing different substituents). All three sites are important for COX-2 binding while sites B and C are important for COX-1 binding. For COX-2 selectivity, only site C plays an important role. The study shows the utility of E-state index in developing statistically acceptable model having direct physicochemical significance.     

QSAR of adenosine receptor antagonists. Part 3: Exploring physicochemical requirements for selective binding of 1,2,4-triazolo[5,1-i]purine derivatives with human adenosine A3 receptor subtype

Considering potential of selective adenosine A3 receptor antagonists in the development of prospective therapeutic agents, an attempt has been made to explore selectivity requirements of 1,2,4-triazolo[5,1-i]purine derivatives for binding with cloned human adenosine A3 receptor subtype. In this study, partition coefficient (logP) values of the molecules (calculated by Crippen's fragmentation method) and Wang-Ford charges of the common atoms of the triazolopurine nucleus (calculated from molecular electrostatic potential surface of energy minimized geometry using AM1 technique) were used as independent variables along with suitable dummy parameters. The best equation describing A3 binding affinity [n=29, Q2=0.796, Ra2=0.853, R2=0.874, R=0.935, s=0.342, F=41.5 (df 4,24), SDEP=0.396] showed parabolic relation with logP (optimum value being 4.134). Further, it was found that an aromatic substituent conjugated with the triazole nucleus should be present at R2 position for A3 binding affinity. Again, high negative charges on N2 and N4 are conducive to the binding affinity. While exploring selectivity requirements of the compounds for binding with A3 receptor over that with A2A receptor, the selectivity relation [n=23, Q2=0.909, Ra2=0.918, R2=0.933, R=0.966, s=0.401, F=62.4 (df 4,18), SDEP=0.412] showed that an aromatic R2 substituent conjugated with the triazole nucleus contributes significantly to the selectivity. Again, presence of a 4-substituted-phenyl ring (except 4-OH-phenyl and 4-CH3-phenyl) at R2 position also increases selectivity. Further, charge difference between N2 and N11 (negative charge on the former should be higher and that on the latter should be less) contributes significantly to the selectivity. In addition, negative charge on N7 is conducive while presence of substituents like propyl, butyl, pentyl or phenyl at R1 position is detrimental for the A3 selectivity.     

Photocatalytic actions of the pesticide metabolite 2-hydroxyquinoxaline: destruction of antioxidant vitamins and biogenic amines - implications of organic redox cycling

Toxicity of the pesticide quinalphos may comprise secondary, delayed effects by its main metabolite 2-hydroxyquinoxaline (HQO). We demonstrate that HQO can destroy photocatalytically vitamins C and E, catecholamines, serotonin, melatonin, the melatonin metabolite AMK (N(1)-acetyl-5-methoxykynuramine), and unsubstituted and substituted anthranilic acids when exposed to visible light. In order to avoid HQO-independent ascorbate oxidation by light and to exclude actions by hydroxyl radicals, experiments on this vitamin were carried out in ethanolic solutions. Other substances tested (vitamin E, melatonin, anthranilic acids) were also photocatalytically destroyed by HQO in ethanol. After product analyses had indicated that HQO was not, or only poorly, degraded in the light, despite its catalytic action on other compounds, we followed directly the time course of HQO and ascorbate concentrations in ethanol. While ascorbate was largely destroyed, no change in HQO was demonstrable within 2 h of incubation. Destruction was not prevented by the singlet oxygen quencher DABCO. Obviously, HQO is capable of undergoing a process of organic redox cycling, perhaps via an intermediate quinoxaline-2-oxyl radical. Health problems from HQO intoxication may not only arise from the loss of valuable biomolecules, such as antioxidant vitamins and biogenic amines, but also from the formation of potentially toxic products. Dimerization and oligomerization are involved in several oxidation processes catalyzed by HQO, especially in the indoleamines, in dopamine, and presumably also in vitamin E. Melatonin oxidation by HQO did not only lead to the well-known - and usually protective - metabolite AFMK (N(1)-acetyl-N(2)-formyl-5-methoxykynuramine), but also to a high number of additional products, among them dimers and trimers. DABCO did not prevent melatonin destruction, but changed the spectrum of products. Serotonin was preferentially converted to a dimer, which can further oligomerize. Several indole dimers are known to be highly neurotoxic, as well as oxidation products formed from catecholamines via the adrenochrome/noradrenochrome pathway. Destruction of melatonin may cause deficiencies in circadian physiology, in immune functions and in antioxidative protection.     

Extra-pituitary action of gonadotropin releasing hormone: possible application

Chronic administration of a potent gonadotropin releasing hormone inhibits ovulation in women. The suppression of gonadal function during long term treatment with the GnRH analogues is ascribable to inhibition of gonadotropin secretion caused by the down regulatory action of the decapeptide at the pituitary level. Reduced progesterone production with premature onset of menstruation has been observed in women injected with the agonist during the midluteal phase. The decapeptide however, has no effect onin vitro human ovarian steroidogenesis. Specific receptors for GnRH have been located on rodent ovarian cells, but corpora lutea of rhesus monkey and human ovaries seem to lack these receptors. The luteolytic effect in women thus appears to be central in origin and not a direct effect on the corpus luteum. Recently, a superactive agonist of GnRH given around the peri-implantation period has been shown to terminate pregnancy in baboons. Monoclonal antibodies against GnRH administered during the same period in a fertile cycle also abrogated pregnancy in these animals. Using immuno-enzymatic techniques GnRH has been localized on the placenta. GnRH also exerts a stimulatory effect on hCG production by the placental villi maintained in culture. Addition of anti-luteinizing hormone releasing hormone antibodies blocks this effect completely. It seems that placenta is the only other tissue besides the pituitary where GnRH has probably a regulatory role in the human female.     

The influence of different 5-methoxyindoles on the process of protein/peptide secretion characterized by the formation of granular vesicles in the mouse pineal gland

Summary The effects of different 5-methoxyindoles on the process of protein/peptide secretion characterized by the formation of granular vesicles (GV) have been studied in mouse pinealocytes maintained in explant culture.All 5-methoxyindoles studied clearly influence the number of granular vesicles in the pinealocytes. Comparing all present results it appeared that, in this system, melatonin was the least effective of all 5-methoxyindoles tested in stimulating secretion. 5-Methoxyindole-3-acetic acid, irrespective of the duration of the experiment and of the presence of noradrenaline, increased the number of GV. For all other 5-methoxyindoles, 5-methoxytryptamine, 5-methoxytryptophan, 5-methoxytryptophol and melatonin, it appeared that the effects depend on the duration of application and on the presence or absence of noradrenaline in the medium. Moreover, depending on the experimental conditions and the 5-methoxyindole tested, antagonistic as well as synergistic effects between 5-methoxyindoles and noradrenaline were observed.The present results, which suggest that the 5-methoxyindoles are also active in the pineal gland itself, demonstrate that, as far as the formation of granular vesicles is concerned, there exists a very complex mechanism of regulation, involving the sympathetic innervation and the 5-methoxyindoles (which are themselves under the influence of this innervation). The physiological significance of this system is discussed in relation to a proposed working hypothesis.IBRO/UNESCO fellow     

Solution structure of DNA/RNA hybrid duplex with C8-propynyl 2'-deoxyadenosine modifications: Implication of RNase H and DNA/RNA duplex interaction

Solution structures of DNA/RNA hybrid duplexes, d(GCGCA*AA*ACGCG): r(cgcguuuugcg)d(C) (designated PP57), containing two C8-propynyl 2'-deoxyadenosines (A*) and unmodified hybrid (designated U4A4) are solved. The C8-propynyl groups on 2'-deoxyadenosine perturb the local structure of the hybrid duplex, but overall the structure is similar to that of canonical DNA/RNA hybrid duplex except that Hoogsteen hydrogen bondings between A* and U result in lower thermal stability. RNase H is known to cleave RNA only in DNA/RNA hybrid duplexes. Minor groove widths of hybrid duplexes, sugar puckerings of DNA are reported to be responsible for RNase H mediated cleavage, but structural requirements for RNase H mediated cleavage still remain elusive. Despite the presence of bulky propynyl groups of PP57 in the minor groove and greater flexibility, the PP57 is an RNase H substrate. To provide an insight on the interactions between RNase H and substrates we have modeled Bacillus halodurans RNase H-PP57 complex, our NMR structure and modeling study suggest that the residue Gly(15) and Asn(16) of the loop residues between first beta sheet and second beta sheet of RNase HI of Escherichia coli might participate in substrate binding.     

Copper accumulation,localization and antioxidant response in <Emphasis Type="Italic">Eclipta alba</Emphasis> L. in relation to quantitative variation of the metal in soil

Eclipta alba L. is a well known medicinal herb, found commonly on contaminated roadsides in Kerala, India. To assess its potential for copper tolerance and accumulation, pot culture experiment was carried out. Metal accumulation in the plant in relation to 50–800 mg kg^?1 Cu in soils, administered as CuSO₄·7H₂O in solution, was examined. Biomass yield of shoot and root, pigment content, Cu accumulation in the plant, bio-concentration factor, and translocation factor were the parameters studied. At the highest level of treatment, Cu was found accumulated more in the roots than in shoots. A significant increase in lipid peroxidation, proline content, phenolics and flavanoids were observed in Cu treated plants, compared to the control. The activity of antioxidant enzymes like superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase was found significantly changed in all the treated plants than in the control. The Bradford assay revealed a significant increase in protein content of the plant at higher levels of Cu treatment. Transmission electron microscopy, images supported the uptake and sequestration of metal particles inside the plant cell. The overall data suggests Eclipta alba L. to be a plant with high potential to tolerate Cu toxicity in soil.     

Modeling the paramyxovirus hemagglutinin-neuraminidase protein

The paramyxovirus hemagglutinin-neuraminidase (HN) protein exhibits neuraminidase activity and has an active site functionally similar to that in influenza neuraminidases. Earlier work identified conserved amino acids among HN sequences and proposed similarity between HN and influenza neuraminidase sequences. In this work we identify the three-dimensional fold and develop a more detailed model for the HN protein, in the process we examine a variety of protein structure prediction methods. We use the known structures of viral and bacterial neuraminidases as controls in testing the success of protein structure prediction and modeling methods, including knowledge-based threading, discrete three-dimensional environmental profiles, hidden Markov models, neural network secondary structure prediction, pattern matching, and hydropathy plots. The results from threading show that the HN protein sequence has a 6 β-sheet propellor fold and enable us to assign the locations of the individual β-strands. The three-dimensional environmental profile and hidden Markov model methods were not successful in this work. The model developed in this work helps to understand better the biological function of the HN protein and design inhibitors of the enzyme and serves as an assessment of some protein structure prediction methods, especially after the x-ray crystallographic solution of its structure. Proteins 29:264–281, 1997. © 1997 Wiley-Liss, Inc.     

Structural insights into the substrate specificity and activity of ervatamins, the papain-like cysteine proteases from a tropical plant, Ervatamia coronaria

Multiple proteases of the same family are quite often present in the same species in biological systems. These multiple proteases, despite having high homology in their primary and tertiary structures, show deviations in properties such as stability, activity, and specificity. It is of interest, therefore, to compare the structures of these multiple proteases in a single species to identify the structural changes, if any, that may be responsible for such deviations. Ervatamin-A, ervatamin-B and ervatamin-C are three such papain-like cysteine proteases found in the latex of the tropical plant Ervatamia coronaria, and are known not only for their high stability over a wide range of temperature and pH, but also for variations in activity and specificity among themselves and among other members of the family. Here we report the crystal structures of ervatamin-A and ervatamin-C, complexed with an irreversible inhibitor 1-[l-N-(trans-epoxysuccinyl)leucyl]amino-4-guanidinobutane (E-64), together with enzyme kinetics and molecular dynamic simulation studies. A comparison of these results with the earlier structures helps in a correlation of the structural features with the corresponding functional properties. The specificity constants (k(cat)/K(m)) for the ervatamins indicate that all of these enzymes have specificity for a branched hydrophobic residue at the P2 position of the peptide substrates, with different degrees of efficiency. A single amino acid change, as compared to ervatamin-C, in the S2 pocket of ervatamin-A (Ala67-->Tyr) results in a 57-fold increase in its k(cat)/K(m) value for a substrate having a Val at the P2 position. Our studies indicate a higher enzymatic activity of ervatamin-A, which has been subsequently explained at the molecular level from the three-dimensional structure of the enzyme and in the context of its helix polarizibility and active site plasticity.     

Unusual fluorescence of W168 in Plasmodium falciparum triosephosphate isomerase, probed by single-tryptophan mutants.

Plasmodium falciparum triosephosphate isomerase (PfTIM) contains two tryptophan residues, W11 and W168. One is positioned in the interior of the protein, and the other is located on the active-site loop 6. Two single-tryptophan mutants, W11F and W168F, were constructed to evaluate the contributions of each chromophore to the fluorescence of the wild-type (wt) protein and to probe the utility of the residues as spectroscopic reporters. A comparative analysis of the fluorescence spectra of PfTIMwt and the two mutant proteins revealed that W168 possesses an unusual, blue-shifted emission (321 nm) and exhibits significant red-edge excitation shift of fluorescence. In contrast, W11 emits at 332 nm, displays no excitation dependence of fluorescence, and behaves like a normal buried chromophore. W168 has a much shorter mean lifetime (2.7 ns) than W11 (4.6 ns). The anomalous fluorescence properties of W168 are abolished on unfolding of the protein in guanidinium chloride (GdmCl) or at low pH. Analysis of the tryptophan environment using a 1.1-A crystal structure established that W168 is rigidly held by a complex network of polar interactions including a strong hydrogen bond from Y164 to the indole NH group. The environment is almost completely polar, suggesting that electrostatic effects determine the unusually low emission wavelength of W168. To our knowledge this is a unique observation of a blue-shifted emission from a tryptophan in a polar environment in the protein. The wild-type and mutant proteins show similar levels of enzymatic activity and secondary and tertiary structure. However, the W11F mutation appreciably destabilizes the protein to unfolding by urea and GdmCl. The fluorescence of W168 is shown to be extremely sensitive to binding of the inhibitor, 2-phosphoglycolic acid.