Diagnosis of enzyme inhibition based on the degree of inhibition

In this work, a method for the diagnosis of kinetic inhibition, based on the dependence of the degree of inhibition (epsilon(i)) on the inhibitor concentration [I] and on the substrate concentration [S], is presented. Because the degree of inhibition is a ratio between rates, kinetic data are normalized by the introduction of an internal control-the rate of the uninhibited reaction. Therefore, the error associated with the kinetic measurements decreases and less experimental measurements are necessary to achieve the diagnosis. The process described, which uses graphical and/or non-linear fitting procedures, allows distinguishing between 20 different kinds of inhibition, including not only linear and hyperbolic, but also parabolic and rational 2,2 inhibitions. Rational 2,2 indicates a new type of inhibition corresponding to an incomplete parabolic inhibition, i.e. mechanistically it corresponds to an inhibitor that binds to two inhibition sites producing enzymatic complexes that are still active. In spite of its comprehensiveness, the diagnosis process is greatly facilitated by the division of the diagnosis of the inhibition in a step-by-step procedure, where only two rival models are evaluated in each step. In the non-linear fittings, the choice between rival models uses a test based on information statistics theory, the Akaike information criterion test, in order to penalize complex models that tend to be favoured in fittings. Finally, equations that allow the determination of inhibition kinetic constants were also deduced. The formalism presented was tested by examining inhibition of acid phosphatase by phosphate (a linear competitive inhibitor).     

Perisomatic inhibition

Recent evidence supports the hypothesis of a functional dichotomy of perisomatic inhibition in the cerebral cortex: the parvalbumin- and cholecystokinin-containing basket cells that are specialized to control rhythm (as a clockwork) and "mood" (as a fine-tuning device), respectively, of network oscillations. Pathology extends this conclusion further, as the former is implicated in epilepsy and the latter in anxiety. The well-balanced cooperation of the two inhibitory systems is required for the normal network operations underlying the cognitive functions of the cerebral cortex.     

Gamma-secretase inhibition

Development of Alzheimer's disease (AD) pathology appears to be causally related to age-dependent changes in the metabolism of the amyloid-beta peptide (A beta), leading to its enhanced aggregation and deposition. gamma-Secretase is a crucial enzyme for the generation of A beta from the amyloid-beta precursor protein and thus represents a valid potential therapeutic target for the treatment or prevention of AD. Enzyme activity has been shown to be dependent on the expression of presenilins and the identification of inhibitors containing transition-state analogue mimics, together with mutagenesis and knockout studies, confirms that presenilins may provide at least a component of the catalytic site for this putative aspartyl protease. Considerable effort has been expended to identify compounds which specifically reduce gamma-secretase activity in the central nervous system, and those with the appropriate properties are being utilized in on-going proof-of-concept studies in animals and humans, to determine the extent and duration of gamma-secretase inhibition required to elicit therapeutic benefits. gamma-Secretase-mediated substrate cleavage appears to fall into the category of 'regulated intramembrane proteolysis'. By virtue of its mechanistic similarities, the effects of gamma-secretase inhibitors on proteolysis and signalling through other substrates, such as Notch, has to be determined carefully, since this is likely to impact on the clinically safe dose of these compounds.     

Low oxygen inhibition of photosynthesis is caused by inhibition of starch synthesis

Photosynthesis of C₃ plants is occasionally inhibited upon switching from normal to low partial pressure of O₂. Leaves of Solanum tuberosum exhibited this effect reproducibly under saturating light and 700 microbars of CO₂. We determined the partitioning of recent photosynthate between starch and sucrose and measured the concentration of hexose monophosphates in the stroma and cytosol after nonaqueous fractionation. The reduction in the rate of photosynthesis upon switching to low partial pressure of O₂ was caused by reduced starch synthesis. The concentration of hexose monophosphates in the stroma fell and the glucose 6-phosphate to fructose 6-phosphate to fructose 6-phosphate ratio fell from 2.7 to 1.3, indicating an inhibition of phosphoglucoisomerase as described by K-J Dietz ([1985] Biochim Biophys Acta 839: 240-248). The concentration of hexose monophosphates in the cytosol increased, ruling out a sucrose synthesis limitation by reduced transport from the chloroplast as the explanation for low O₂ inhibition of photosynthesis.     

Lithium inhibition of phosphofructokinase

The mode of action of lithium in prophylaxis of recurrent affective disorder is unknown although it has been suggested that lithium might compete with magnesium in magnesium dependent processes. We have previously shown pyruvate kinase to be inhibited by lithium and the present study demonstrates a small inhibition by lithium of phosphofructokinase that is also a major regulatory step in glycolysis. Inhibition of PFK was competitive with respect to ATP and magnesium and noncompetitive with respect to potassium and fructose-6-phosphate. Inhibition was enhanced at reduced concentrations of magnesium.     

Allosteric inhibition of BCR-ABL

Impaired regulation of kinase activity can lead to a variety of diseases, including cancer. Inhibition of kinase activity has, therefore, been considered an attractive anti-cancer therapeutic strategy. The success of targeted therapy with kinase inhibitors has been well documented with BCR-ABL, where imatinib specifically inhibits kinase activity with impressive pharmacological responses in chronic myelogenous leukemia (CML). However, the success of kinase inhibitors as cancer therapeutics is being challenged clinically by the emergence of acquired resistance. Most kinase inhibitors available today are ATP-competitive. There have been efforts to develop kinase inhibitors with new modes of action. In this review, we highlight the development of 'allosteric kinase inhibitors' that inhibit kinase activity by binding to a site remote from the active site of the kinase. We focus on recent efforts directed towards BCR-ABL, for which, significant progress has been made to develop allosteric inhibitors with promising therapeutic activity, especially in the context of overcoming clinically acquired resistance mutations to the first generation of ATP-competitive kinase inhibitors.     

Oxygen inhibition of photosynthesis

The effect of leaf temperature, O₂ and calculated O₂/CO₂ solubility ratio in the leaf on the quantum yield of photosynthesis was studied for the C₄ species, Zea mays L., and the C₃ species, Triticum aestivum L. Over a range of leaf temperatures of 16 to 35° C, the quantum yield of Z. mays was relatively constant and was similar under 1.5 and 21% O₂, being ca. 0.059 mol CO₂ mol^-1 quanta absorbed. Under 1.5% O₂ and atmospheric levels of CO₂, the quantum yield of T. aestivum was relatively constant (0.083 mol CO₂ mol^-1 quanta absorbed) at leaf temperatures from 15 to 35° C. Atmospheric levels of O₂ (21%) reduced the quantum yield of photosynthesis in T. aestivum and as leaf temperature increased, the quantum yield decreased from 0.062 at 15°C to 0.046 mol CO₂ mol^-1 quanta absorbed at 35°C. Increasing temperature decreases the solubility of CO₂ relatively more than the solubility of O₂, resulting in an increased solubility ratio of O₂/CO₂. Experimentally manipulating the atmospheric levels of O₂ or CO₂ to maintain a near-constant solubility ratio of O₂/CO₂ at varying leaf temperatures largely prevented the temperature-dependent decrease in quantum yield in t. aestivum. Thus, the decrease in quantum yield with increasing leaf temperature in C₃ species may be largely caused by a temperaturedependent change in the solubility ratio of O₂/CO₂.J and II=Ku and Edwards, 1977a, b     

Bacterial inhibition of phagocytosis

The concerted study of molecular mechanisms of phagocytosis and the inhibition of phagocytosis by specific products of extracellular bacterial pathogens has borne considerable fruit. The importance of tyrosine phosphorylation and of the Rho family of GTPases has become clear to cell biologists, but pathogenic bacteria recognized the importance of these signalling pathways in phagocytic cells long ago. The discoveries described in this review are only the beginning. The simultaneous pursuit of the mechanisms and molecules involved in the initiation and regulation of phagocytosis and that pathogenic bacteria use to inhibit phagocytosis will surely identify more interesting pathways on each side of the contest. Are there any obvious possibilities? There are several bacterial factors that have the potential to inhibit known mechanisms of phagocytosis. Clostridium species, for example, make a number of exotoxins of interest. Clostridium botulinum and Clostridium tetani neurotoxins inactivate the regulated secretory machinery by proteolytic cleavage of SNARE proteins, and targets of tetanus toxin and botulinum b toxin inhibit the exocytotic delivery of membrane vesicles needed for phagocytosis of large particles (Hackam et al., 1998). Moreover, the C3 exotoxin of C. botulinum catalyses ADP ribosylation and inactivation of rho family GTPases (Wiegers et al., 1991), and toxins A and B of C. difficile UDP-glucosylate and inactivate rho GTPases and thereby disrupt the actin cytoskeleton (Just et al., 1995a,b). However, as Clostridia lack the machinery for type III secretion, these proteins are not rapidly targeted to the phagocyte cytoplasm. More searching may reveal a pathogen that has combined the type III secretory machinery with clostridia toxin-like substrates. A potentially unique strategy for remaining outside phagocytes is exhibited by Helicobacter pylori, which contain a type IV secretion system. Unopsonized virulent strains of H. pylori bind readily to macrophages but are only internalized after a delay of several minutes. Such a delay appears to be sufficient for the bacteria to remain extracellular (Allen et al., 2000). Elucidation of the mechanism used by H. pylori to delay phagocytosis may reveal one or more novel virulence factors as well as one or more novel targets in the phagocyte that will add to the understanding of a fundamental process in host defence. Another field ripe for further mechanistic investigation is complement receptor-mediated phagocytosis. Dedicated study of the molecular events and molecular mediators of phagocytosis downstream of CR3 is likely to reveal interesting differences from FcgammaR phagocytosis and is just as likely to reveal that microbes have discovered unique mechanisms for circumventing them. Study of extracellular pathogens and the mechanisms that they use to remain outside phagocytic cells has revealed a great deal about the initial encounter between pathogen and phagocyte. We can look forward to additional discoveries about the host-pathogen interactions and the mechanisms and factors that each side uses to battle against the other.     

Immunological inhibition of carcinogenesis

The combination of new information provided by fundamental immunology, along with the refinement of genetic engineering techniques has given scientists the capacity to produce vaccines able to inhibit the growth of most if not every transplantable tumor. However, when faced with already established tumors, vaccines fail to afford any significant protection. Many studies are underway which seek to overcome this gloomy situation. However, another possibility is to follow the indications provided by a large quantity of experimental data and to evaluate the possibility of using immunotherapy to prevent the initial stages of tumor growth. Is it possible to prevent an autologous tumor by means of a vaccination performed before tumor onset? Could antitumor vaccines be a new form of preventive medicine in the wake of Jenner, Pasteur, and other pioneers? In this paper it is our intention to review the results obtained by our laboratory in the attempt to use natural and adaptive immunity in the control of carcinogenesis. Natural immunity boosted by IL-12 and IL-2 significantly hampers the progression of mammary lesions occurring in HER-2/neu transgenic mice genetically predestined to develop lethal mammary carcinomas. Specific immunity elicited by DNA vaccination provides a much stronger inhibition of the development of mammary lesions, and a significant number of transgenic mice are tumor free at 1 year of age. These experimental data suggest the possibility of using immunity as a means of controlling preneoplastic lesions and protecting healthy persons at risk of developing cancer.