Metabolic analysis in drug design

Biotechnology is often presented as if progress in the past two decades represented a major success, but the reality is quite different. For example, ten major classes of antibiotics were discovered between 1935 and 1963, but after 1963 there has been just one, the oxazolidones. To illustrate the possibilities of doing better by taking account of the real behaviour of metabolic systems, we can examine how one might modify the activity of an enzyme in the cell (for example by genetic manipulation, or by the action of an inhibitor, etc.) to satisfy a technological aim. For example, if the objective is to eliminate a pest, one might suppose that the effect of an inhibitor could be to depress an essential flux to a level insufficient for life, or to raise the concentration of an intermediate to a toxic level. The former may seem the more obvious, but the latter is easier to achieve in practice, and there are some excellent examples of industrial products that work in that way, such as the herbicide 'Roundup' and antimalarials of the quinine class. A study of glycolysis in the parasite Trypanosoma brucei (which causes African sleeping sickness) indicates that for this approach to work the selected target enzyme must have a substrate with a concentration that is not limited by stoichiometric constraints. That is not necessarily easy to find in a complicated system, and typically needs the metabolic network to be analysed in the computer.     

Binding of a tritiated enkephalinergic analog (FK 33-824) to a mitochondrial fraction of rat brain

FK-33-824 (Try-D-Ala-Gly-MePhe-Met(O)ol) is a potent enkephalin analog which has been tritium labelled with a high specific radioactivity (41 Ci/mmole). The labelled drug exhibits specific and saturable binding to rat brain crude mitochondrial fraction. Specific binding is inhibited by low concentrations of morphine, levallorphan and beta-endorphin, suggesting that FK 33-824 [3H] binds preferentially to mu opiate sites. Binding studies at equilibrium and kinetics of formation and dissociation of the labelled ligand-receptor complex indicate that FK 33-824 [3H] binds to two classes of specific sites. Their affinities are distinguishable at 0 degree (KD = 1.3 and 5.8 nM) and very close to each other at 37 degree (KD = 1.9 nM).     

Sunflower seed protein: size and charge heterogeneity in subunits of the globulin fraction

The subunit heterogeneity of the globulin fraction of sunflower seeds was investigated by two dimensional electrophoresis, using isoelectric focusing in the first dimension and sodium dodecyl sulphate polyacrylamide gel electrophoresis in the second dimension. Under non reducing conditions, intermediary subunits B, C and D (molecular weight 54 000, 48 000 and 40 000, respectively) were focused within a pI range 5.4-6.0 but intermediary subunits A (molecular weight 60 000) focused within a pI range 6.3-6.8. Under reducing conditions the electrophoretic patterns show that intermediary subunits consist in large "acidic" and small "basic" subunits linked by disulphide bonds. The large subunits of B species are more acidic and less heterogeneous than the corresponding subunits of the A species. These results confirm that helianthinin had a "legumin-type" structure.     

Ecologie des Dermestidae,une famille de Coléoptères nécrophages associée aux cadavres squelettisés

Lorsqu’un animal meurt, son cadavre forme un écosystème local instable et éphémère où différentes communautés d’insectes nécrophages vont se nourrir. Cette étude a pour but de caractériser les relations entre deux familles fréquentes en contextes médico-légal : les Diptères Calliphoridae et les Coléoptères Dermestidae. Des expériences de choix binaire (olfactomètres en T) et de prédation des Dermestes sur les larves de Diptères ont constitué le 1er axe de notre étude. Il en ressort que l’espèce D. maculatus (adultes et larves) n’exerce qu’une très faible prédation sur les larves de L. sericata. En absence de nourriture, les Dermestes sont néanmoins capables de prédater les pupes de L. sericata, et donc de faire disparaitre ces traces particulièrement importantes en entomologie médico-légale et en archéo-entomologie. Le second axe s’est focalisé sur les relations de compétition et d’agrégation entre D. maculatus et D. haemorrhoidalis. Les résultats obtenus n’ont pas mis en évidence de mécanismes de compétition ou d’exclusion entre ces deux espèces pourtant amenées à fréquenter simultanément le même habitat et à exploiter les mêmes ressources. Placées en condition de choix binaire (2 spots identiques), les 2 espèces s’agrègent ensemble. Mais le développement conjoint de ces 2 espèces sur une seule source de nourriture non limitante entraine une baisse significative du taux de survie chez les deux espèces. Au vu de ces résultats, nous avons conclu à la possibilité d’une coexistence de ces différentes espèces (L. sericata, D. maculatus et D. haemorrhoidalis) sur un cadavre.     

Long-lasting H-reflex inhibition evoked by stimulation of a nerve to the antagonist muscles and vibrational stimulation of the muscle receptors in humans

In testing of healthy subjects, we studied the influence of conditioning stimulation of then.peroneus communis on the H-reflex EMG discharge recorded from them. soleus. Two waves of inhibition separated by a period of relative facilitation of the reflex were observed. The first wave,D ₁, was not longer than 50 msec. The intensity of later inhibition (D ₂ wave) within a 500 to 4000 msec time segment linearly depended on the logarithm of the interval between the conditioning and test stimuli. Extrapolation showed that the duration of this wave could be evaluated as equal to 6.3 msec, on average. We also studied the temporal course of H-reflex inhibition after conditioning vibrational stimulation of the receptors of them. tibialis ant. andm. biceps femori. Within a 500 to 4000 msec test interval, it could also be fitted with a logarithmic dependence similar to that observed after electrical stimulation of then. peroneus comm. The duration of inhibition after vibrational stimulation of them. tibialis ant. andm. biceps femori was 6.2 and 8.9 sec, respectively. Inhibition evoked by both conditioning influences was not removed after a voluntary plantar flexion of the foot. Our observations support the statement that in humans stimulation of the afferent fibers from the antagonist muscles, as well of the muscle receptors, evokes in two-neuron reflex arcs presynaptic inhibition, whose duration reaches several seconds; this phenomenon can play a significant role in the control of muscle tone in the course of muscle performance.     

Kinetic behaviour of proenzymes activation in the presence of different inhibitors for both activating and activated enzymes

In the present paper, a kinetic analysis of a general model for proenzyme activation, where the activating enzyme and also the activated one are reversibly inhibited in two steps by two different inhibitors, has been performed. The cases in which both inhibitors are the same, or in which the inhibition is irreversible (only one or the two inhibition routes) are treated as particular cases of the general model. In addition, the kinetic behaviour of many other proenzyme activation systems involving inhibition, particular cases of the reaction scheme under study, can be obtained. The total number of particular cases for the general model under study is 370, so this approach offers to the scientific community working in limited proteolysis regulation for the first time a method based on general solutions which only needs to be specified to their concrete problem of zymogen activation. Finally, new adimensional parameters are introduced, allowing the knowledgement, in the case that any of the inhibition routes is irreversible, the relative weight of both activation and irreversible inhibition routes.     

The inhibition effect of some n-alkyl dithiocarbamates on mushroom tyrosinase

Three new n-alkyl dithiocarbamate compounds, as sodium salts, C₄H₉NHCS₂Na (I), C₆H₁₃NHCS₂Na (II) and C₈H₁₇NHCS₂Na (III), were synthesized and examined for inhibition of both cresolase and catecholase activities of mushroom tyrosinase (MT) from a commercial source of Agaricus bisporus in 10 mM phosphate buffer pH 6.8, at 293K using UV spectrophotometry. Caffeic acid and p-coumaric acid were used as natural substrates for the enzyme for the catecholase and cresolase reactions, respectively. Lineweaver–Burk plots showed different patterns of mixed and competitive inhibition for catecholase and cresolase reactions, respectively. These new synthetic compounds can be classified as potent inhibitors of MT due to K_i values of 0.8, 1.0 and 1.8 μM for cresolase inhibitory activity, and also 9.4, 14.5 and 28.1 μM for catecholase inhibitory activity for I, II and III, respectively. They showed a greater potency in the inhibitory effect towards the cresolase activity of MT. Both substrate and inhibitor can be bound to the enzyme with negative cooperativity between the binding sites (α>1) and this negative cooperativity increases with increasing length of the aliphatic tail in these compounds. The inhibition mechanism is presumably related to the chelating of the binuclear coppers at the active site and the different K_i values may be related to different interaction of the aliphatic chains of I, II and III with the hydrophobic pocket in the active site of the enzyme.     

The inhibitory effect of some new synthesized xanthates on mushroom tyrosinase activities

Three iso-alkyldithiocarbonates (xanthates), as sodium salts, C₃H₇OCS₂Na (I), C₄H₉OCS₂Na (II) and C₅H₁₁OCS₂Na (III), were synthesized, by the reaction between CS₂ with the corresponding iso-alcohol in the presence of NaOH, and examined for inhibition of both cresolase and catecholase activities of mushroom tyrosinase (MT) from a commercial source of Agricus bisporus. 4-[(4-methylbenzo)azo]-1,2-benzendiol (MeBACat) and 4-[(4-methylphenyl)azo]-phenol (MePAPh) were used as synthetic substrates for the enzyme for the catecholase and cresolase reactions, respectively. Lineweaver-Burk plots showed different patterns of mixed and competitive inhibition for the three xanthates and also for cresolase and catecholase activities of MT. For cresolase activity, I and II showed a mixed inhibition pattern but III showed a competitive inhibition pattern. For catecholase activity, I showed mixed inhibition but II and III showed competitive inhibition. These new synthesized compounds are potent inhibitors of MT with K_i values of 9.8, 7.2 and 6.1 μM for cresolase inhibitory activity, and also 12.9, 21.8 and 42.2 μM for catecholase inhibitory activity for I, II and III, respectively. They showed a greater inhibitory potency towards the cresolase activity of MT. Both substrate and inhibitor can be bound to the enzyme with negative cooperativity between the binding sites (α>1) and this negative cooperativity increases with increasing length of the aliphatic tail in these compounds in both cresolase and catecholase activities. The cresolase inhibition is related to the chelating of the copper ions at the active site by a negative head group (S^? ) of the anion xanthate, which leads to similar values of K_i for all three xanthates. Different K_i values for catecholase inhibition are related to different interactions of the aliphatic chains of I, II and III with hydrophobic pockets in the active site of the enzyme.     

In vitro inhibition of fatty acid synthase by 1,2,3,4,6-penta-O-galloyl-β-d-glucose plays a vital role in anti-tumour activity

1,2,3,4,6-Penta-O-galloyl-β-d-glucose (PGG) inhibits glioma cancer U251 cells, more strongly than MDA-MB-231 and U87 cells. In addition, PGG is transported across cancer cell membrane to further down-regulate FAS and activate caspase-3 in MDA-MB-231 cells. Compared with other FAS inhibitors, including catechin gallate and morin, PGG involves a higher reversible fast-binding inhibition with half-inhibitory concentration value (IC₅₀) of 1.16 μM and an irreversible slow-binding inhibition, i.e. saturation kinetics with a dissociation constant of 0.59 μM and a limiting rate constant of 0.16 min^−l. The major reacting site of PGG is on the β-ketoacyl reduction domain of FAS. PGG exhibits different types of inhibitions against the three substrates in the FAS overall reaction. The higher concentrations of PGG tested (higher than 20 μM) clearly altered the secondary structure of FAS by increasing the α-helix and induced a redshift in the FAS spectra. In addition, only PGG concentrations higher than 20 μM resulted in FAS precipitation.     

Regulation of glucose 6-phosphate dehydrogenase in Zymomonas mobilis CP4

Abstract Glucose 6-phosphate dehydrogenase was purified 29-fold from Zymomonas mobilis . The enzyme was active with both NAD and NADP. Phosphoenolpyruvate was found to be a negative allosteric effector and ATP inhibited the enzyme non-allosterically, at physiological concentrations.     

Status of the so-called African pygmy elephant (Loxodonta pumilio (NOACK 1906)): phylogeny of cytochrome b and mitochondrial control region sequences

Among the African elephants, it has been unanimously acknowledged that the forest elephants (cyclotis form) are peculiar, so that they have been elevated to the specific rank. The development of molecular analyses of extant Loxodonta has only focused on two forms yet: the savannah form (africana) and the forest form (cyclotis), disregarding the so-called pygmy elephants (pumilio or fransseni) the systematic status of which has been debated since their discovery. Therefore, we have sampled nine dwarfed-labelled specimens in collection and eight specimens of typical forest elephants that we compared to three savannah elephants and two Asian elephants. Because of the degraded nature of the nuclear DNA content in bone samples of old specimens, we assayed mitochondrial markers; 1961 bp of the mitochondrial genome were sequenced (over a continuous range spanning the cytochrome b gene, tRNA Thr, tRNA Pro, hypervariable region 1 and central conserved region of the control region). Pumilio and cyclotis are not sister-taxa: the phylogenetic analyses rather account for the inclusion of the so-called pygmy elephants within a monophyletic group of forest elephants sensu lato. The internal structure of this clade reveals to depend on isolation and remoteness between populations, characteristics that may have been extensively influenced by climatic variations during the Quaternary period. We conclude that the specific taxon Loxodonta pumilio (or Loxodonta fransseni) should be abandoned.     

Les brachiopodes carbonifères des Pyrénées centrales françaises: 2: Étude des Orthida et des Spiriferida; biostratigraphie,paléoécologie,paléobiogéographie

This paper follows the first part: Legrand-Blain &alii, 1983,Geobios, n^o 16, fasc. 3.The brachiopods here described from the «Culm are: Schizophoria cf. resupinata, Rhipidomella michelini, Actinoconchus aff. paradoxus, Lamellosathyris gr. lamellosa, Cleiothyridina (Leiothycridina) nov. sp. gr. okensis, Podtsheremia (s.l.) convenarum nov. sp., Neospirifer gr. derjawini, Anthracospirifer (?) sp., Brachythyrina (Anthracothyrina) perextensa nov. subgen., nov. sp., «Spirifer lujkiensis, Zaissania nov. sp., Kitakamithyris or Torynifer (?) sp., Martinia minima. From shales associated with the Ardengost limestones, slightly different faunas are: Podtsheremia (s.l.) convenarum (?), Anthracospirifer (?) sp., Brachythyrina (Anthracothyrina) bressoni nov. subgen., nov. sp., Phricodothyris mosquensis, Martinia buckmani.The brachiopods from Ardengost limestones andassociated shales, well dated, are of upper Serpukhovian age. The «Culm ones might be heterochroneous, with Serpukhovian and (?) Bashkirian elements. The paleoecology of the brachiopods from limestones and associated shales indicates moderate and low energy environments. The «Culm brachiopods display both resedimentation and delicate preservation features. There is evidence of rather close palaeobiogeographic relationships with the northern Sahara: during the late Serpukhovian, a «Sahara-Pyrénées region could be distinguished, at the western part of the vast Tethysian province.