Carbonic anhydrase inhibitors; phosphoryl-sulfonamides--a new class of high affinity inhibitors of isozymes I and II

A series of phosphorylated aromatic/heterocyclic sulfonamides with the general formula ArSO2NHPO3H2 have been prepared by condensing ArSO2NH2 with phosphorus pentachloride, followed by controlled hydrolysis in the presence of formic acid. The new derivatives generally act as stronger inhibitors of two carbonic anhydrase (CA) isozymes, CA I and CA II, as compared to the parent unsubstituted sulfonamides from which they were obtained. The inhibition mechanism by this new class of CA inhibitors, as well as structure activity correlations for the series of investigated derivatives, are also discussed.     

Neuronal interspike time distributions and the estimation of neurophysiological and neuroanatomical parameters

The problem of creating proteins with new functions out of already existing proteins is treated in some detail. It is shown that the generally accepted process of a gene-duplication followed by random mutational events should have been inefficient as a means of evolution in primitive cells. An alternative scheme, based on considerations of protein folding processes, is presented, and is shown, by the means of a specific example, to give a consistent picture of the evolution of primitive proteins with new functions. In this scheme the emergence of the new function occurs prior to the gene-duplication.     

Basic cerebellar events relating sensorimotor adaptation--structural analysis on extracellular records of Purkinje cells

The heterosynaptic facilitation or suppression of synaptic efficacy between parallel fibers and the dendritic spines of Purkinje cells (PCs) in the cerebellar cortex has been for decades the basic idea of sensorimotor adaptation. Great efforts in order to get direct evidence failed, or were not accepted as direct proofs. A new facility was introduced with the structural analysis of intradendritic records of the PC. These records reveal a generally double (rarely single, triple or quadruple) rhythmic pattern of small spikes, which are proposed to be prespikes of dendritic origin. Moreover, they may take their origin at functionally separated dendritic compartments resulting in a nonlinear, phase-sensitive integrative process, performed by the compound spike generating mechanism of the cerebellar PC.     

Comparative physiology and molecular evolution of carbonic anhydrase in the erythrocytes of early vertebrates

The different isozymes of carbonic anhydrase (CA) have been the subject of intensive study in mammals, but there is still much to be learned about the early evolution of this enzyme in vertebrates. Erythrocyte CA plays an essential role in the respiratory processes of most vertebrates and is probably the most well studied CA isozyme. The available evidence indicates that there has been a progressive increase in the efficiency of erythrocyte CA during the early evolution of vertebrates. There also appears to be a substantial increase in erythrocyte CA activity during development in some species. At the present time, however, the selective pressures that may be influencing the properties of erythrocyte CA during vertebrate evolution and development have not been clearly determined. When the available molecular sequence information is examined, it is evident that the erythrocyte CAs of early vertebrates have active sites that are more similar to those of mammalian CA VII and II, rather than CA I. We can now also begin to examine the phylogenetic relationships between the different rbc CAs in vertebrates, but more CA sequence information is clearly required from different groups of vertebrates before we have a complete picture of the molecular evolution of erythrocyte CA.     

Discovering a new class of antifungal agents that selectively inhibits microbial carbonic anhydrases

Infections caused by pathogens resistant to the available antimicrobial treatments represent nowadays a threat to global public health. Recently, it has been demonstrated that carbonic anhydrases (CAs) are essential for the growth of many pathogens and their inhibition leads to growth defects. Principal drawbacks in using CA inhibitors (CAIs) as antimicrobial agents are the side effects due to the lack of selectivity toward human CA isoforms. Herein we report a new class of CAIs, which preferentially interacts with microbial CA active sites over the human ones. The mechanism of action of these inhibitors was investigated against an important fungal pathogen, Cryptococcus neoformans, revealing that they are also able to inhibit CA in microbial cells growing in vitro. At our best knowledge, this is the first report on newly designed synthetic compounds selectively targeting β-CAs and provides a proof of concept of microbial CAs suitability as an antimicrobial drug target.     

Abscisic acid-induced gene-expression requires the activity of protein(s) sensitive to the protein-tyrosine phosphatase inhibitor phenylarsine oxide

Abscisic acid-induced gene-expression requires the activity of protein(s) sensitive to the protein-tyrosine phosphatase inhibitor phenylarsine oxide. It is generally accepted that phosphorylation/dephosphorylation of proteins plays an important role in signal transduction cascades. evidence is now accumulating that for plants the same holds true. To study the role of phosphorylation in ABA signal transduction, we used six different compounds which were reported to inhibit phosphatase action. Three of these inhibitors: phenylarsine oxide (PAO), Calyculin A (CA) and Okadaic Acid (OA) appeared capable of inhibiting ABA-induced gene-expression. The same three inhibitors are shown to bring about hyperphosphorylation of two approximately 40 kDa proteins, present in the membrane-bound fraction of barley aleurone cells. The other three inhibitors had no visible effect on the phosphorylation status of the barley proteins. The hyperphosphorylation of the two 40 kDa proteins coincided with an increase of tyrosine-phosphorylation of two 40 kDa proteins with different pI, as determined with anti-phosphotyrosine antibodies.     

3H-1,2-benzoxathiepine 2,2-dioxides: a new class of isoform-selective carbonic anhydrase inhibitors

A new chemotype with carbonic anhydrase (CA, EC 4.2.1.1) inhibitory action has been discovered, the homo-sulfocoumarins (3H-1,2-benzoxathiepine 2,2-dioxides) which have been designed considering the (sulfo)coumarins as lead molecules. An original synthetic strategy of a panel of such derivatives led to compounds with a unique inhibitory profile and very high selectivity for the inhibition of the tumour associated (CA IX/XII) over the cytosolic (CA I/II) isoforms. Although the CA inhibition mechanism with these new compounds is unknown for the moment, we hypothesize that it may be similar to that of the sulfocoumarins, i.e. hydrolysis to the corresponding sulfonic acids which thereafter anchor to the zinc-coordinated water molecule within the enzyme active site.     

Reflections on sterol sidechain cleavage process catalyzed by cytochrome P450(scc)

The essay examines the evidence upon which the presently accepted version of the mechanism of the cytochrome P450(scc)-catalyzed-cleavage of the sidechain of cholesterol is based. This analysis indicates that the generally held view of the process (two consecutive hydroxylations, followed by cleavage of the resulting glycol) most likely does not describe the true mechanism. The available evidence can not be used to support this traditional notion. Two alternative hypotheses are proposed.     

Discovery of Strecker-type α-aminonitriles as a new class of human carbonic anhydrase inhibitors using differential scanning fluorimetry

A new type of carbonic anhydrase inhibitors was identified via differential scanning fluorimetry (DSF) screening. The compounds displayed interesting inhibition profile against human carbonic anhydrase isoforms I, II, IX and XII with an obvious selectivity displayed by one compound toward carbonic anhydrase (CA) IX, an established anti-cancer target. A hypothetical mechanism of inhibitory action by the Strecker-type α-aminonitriles has been proposed.     

Structure-based drug discovery of carbonic anhydrase inhibitors

Inhibition of the metalloenzyme carbonic anhydrase (CA; EC 4.2.1.1) has pharmacologic applications in the field of anti-glaucoma, anti-convulsant and anti-cancer agents. But recently, it has also emerged that these enzymes have the potential for designing anti-infective drugs (anti-fungal and anti-bacterial agents) with a novel mechanism of action. Sulphonamides and their isosteres (sulphamates/sulphamides) constitute the main class of CA inhibitors (CAIs), which bind to the metal ion from the enzyme active site. Recently, the dithiocarbamates (DTCs), possessing a similar mechanism of action, were reported as a new class of inhibitors. These types of CAIs will be discussed in detail in this review. Novel drug design strategies have been reported ultimately based on the tail approach for obtaining sulphonamides/DTCs, which exploit more external binding regions within the enzyme active site (in addition to coordination to the metal ion), leading thus to isoform-selective compounds. Most of the promising data have been obtained by combining x-ray crystallography of enzyme-inhibitor adducts with novel synthetic approaches for generating chemical diversity. Whereas sulphonamide – NO donating hybrid drugs were reported as effective anti-glaucoma agents, most of the interesting new inhibitors were designed for inhibiting specifically the tumour-associated isoforms CA IX and XII, validated targets for imaging and treatment of hypoxic tumours. Promising compounds that inhibit CAs from bacterial and fungal pathogens, of the DTC and carboxylate types, will be also reviewed.     

Involvement of autophagy in the response of tumor cells to PtdIns3K inhibitors

The phosphoinositide 3-kinase (PI3K) pathway plays a crucial role in cell proliferation and survival and is frequently activated by genetic and epigenetic alterations in human cancer. An arsenal of pharmacological inhibitors of key signaling enzymes in this pathway, including class I_A PI3K isoforms, has been developed in the past decade and several compounds have entered clinical testing in cancer patients. The PIK3CA/p110α isoform is the most studied enzyme of the family and a validated cancer target. The induction of autophagy by PI3K pathway inhibitors has been documented in various cancers, although a clear picture about the significance of this phenomenon is still missing, especially in the in vivo situation. A better understanding of the contribution of autophagy to the action of PI3K inhibitors on tumors cells is important, since it may limit or enhance the action of these compounds, depending on the cellular context.     

A nonlinear dynamic approach reveals a long-term stroke effect on cerebral blood flow regulation at multiple time scales

Cerebral autoregulation (CA) is an important vascular control mechanism responsible for relatively stable cerebral blood flow despite changes of systemic blood pressure (BP). Impaired CA may leave brain tissue unprotected against potentially harmful effects of BP fluctuations. It is generally accepted that CA is less effective or even inactive at frequencies >∼0.1 Hz. Without any physiological foundation, this concept is based on studies that quantified the coupling between BP and cerebral blood flow velocity (BFV) using transfer function analysis. This traditional analysis assumes stationary oscillations with constant amplitude and period, and may be unreliable or even invalid for analysis of nonstationary BP and BFV signals. In this study we propose a novel computational tool for CA assessment that is based on nonlinear dynamic theory without the assumption of stationary signals. Using this method, we studied BP and BFV recordings collected from 39 patients with chronic ischemic infarctions and 40 age-matched non-stroke subjects during baseline resting conditions. The active CA function in non-stroke subjects was associated with an advanced phase in BFV oscillations compared to BP oscillations at frequencies from ∼0.02 to 0.38 Hz. The phase shift was reduced in stroke patients even at > = 6 months after stroke, and the reduction was consistent at all tested frequencies and in both stroke and non-stroke hemispheres. These results provide strong evidence that CA may be active in a much wider frequency region than previously believed and that the altered multiscale CA in different vascular territories following stroke may have important clinical implications for post-stroke recovery. Moreover, the stroke effects on multiscale cerebral blood flow regulation could not be detected by transfer function analysis, suggesting that nonlinear approaches without the assumption of stationarity are more sensitive for the assessment of the coupling of nonstationary physiological signals.     

Detergent-resistant membranes should not be identified with membrane rafts

Three originally distinct concepts - lipid rafts, detergent-resistant membranes (DRMs) and liquid-ordered (lo) lipid phases - are often confused in current literature; many researchers have assumed that all three names refer to the same chemico-biological entity. In fact, theoretical and experimental findings provide strong evidence against identifying DRMs with rafts and lo domains. Because much of what we think we know about lipid rafts is based on their unjustified identification as DRMs, functional domains in biological membranes might differ markedly from the generally accepted picture.     

B染色体起源的研究进展

B染色体(简写为Bs)起源的传统观点是认为它起源于携带者所在物种的基因组.目前又发现了许多新的证据来推测Bs的起源.它可能具有两种起源:种内起源和种间起源(起源于另一物种的基因组),且有证据表明同一物种的Bs可能是多次起源,同时对Bs的起源机制也作了总结.认为B8起源的研究已取得了一定的进展,但要给Bs起源下一个确切的结论仍需要大量的实验证据.     

Carbonic anhydrase inhibitors. Synthesis of 2,4,6-trimethylpyridinium derivatives of 2-(hydrazinocarbonyl)-3-aryl-1H-indole-5-sulfonamides acting as potent inhibitors of the tumor-associated isoform IX and XII

A series of 2-(hydrazinocarbonyl)-3-aryl-1H-indole-5-sulfonamides possessing various 2-, 3- or 4- substituted phenyl groups with methyl-, halogeno- and methoxy-functionalities, or a perfluorophenyl moiety, has been derivatized by reaction with 2,4,6-trimethylpyrylium perchlorate. The new sulfonamides were evaluated as inhibitors of four mammalian carbonic anhydrase (CA, EC 4.2.1.1) isoforms, that is, CA I, II (cytosolic), CA IX and XII (transmembrane, tumor-associated forms). Excellent inhibitory activity was observed against hCA IX with most of these sulfonamides, and against hCA XII with some of the new compounds. These compounds were generally less effective inhibitors of hCA II. Being membrane impermeant, these positively-charged sulfonamides are interesting candidates for targeting the tumor-associated CA IX and XII, as possible diagnostic tools or therapeutic agents.     

Carbonic anhydrase IX: Biochemical and crystallographic characterization of a novel antitumor target

Isoform IX of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1), CA IX, is a transmembrane protein involved in solid tumor acidification through the HIF-1α activation cascade. CA IX has a very high catalytic activity for the hydration of carbon dioxide to bicarbonate and protons, even at acidic pH values (of around 6.5), typical of solid, hypoxic tumors, which are largely unresponsive to classical chemo- and radiotherapy. Thus, CA IX is used as a marker of tumor hypoxia and as a prognostic factor for many human cancers. CA IX is involved in tumorigenesis through many pathways, such as pH regulation and cell adhesion control. The X-ray structure of the catalytic domain of CA IX has been recently reported, being shown that CA IX has a typical α-CA fold. However, the CA IX structure differs significantly from the other CA isozymes when the protein quaternary structure is considered. Thus, two catalytic domains of CA IX associate to form a dimer, which is stabilized by the formation of an intermolecular disulfide bond. The active site clefts and the proteoglycan (PG) domains are located on one face of the dimer, while the C-termini are located on the opposite face to facilitate protein anchoring to the cell membrane. As all mammalian CAs, CA IX is inhibited by several main classes of inhibitors, such as the inorganic anions, the sulfonamides and their bioisosteres (sulfamates, sulfamides, etc.), the phenols, and the coumarins. The mechanism of inhibition with all these classes of compounds is understood at the molecular level, but the sulfonamides and their congeners have important applications. It has been recently shown that both in vitro, in cell cultures, as well as in animals with transplanted tumors, CA IX inhibition with sulfonamides lead to a return of the extracellular pH to more normal values, which leads to a delay in tumor growth. As a consequence, CA IX represents a promising antitumor target for the development of anticancer agents with an alternative mechanism of action.     

Activity-dependent changes of the hippocampal CA3–CA1 synapse during the acquisition of associative learning in conscious mice

Contemporary neuroscientists are paying increasing attention to subcellular, molecular and electrophysiological mechanisms underlying learning and memory processes. Recent efforts have addressed the development of transgenic mice affected at different stages of the learning process, or emulating pathological conditions involving cognition and motor-learning capabilities. However, a parallel effort is needed to develop stimulating and recording techniques suitable for use in behaving mice, in order to grasp activity-dependent neural changes taking place during the very moment of the process. These in vivo models should integrate the fragmentary information collected by different molecular and in vitro approaches. In this regard, long-term potentiation (LTP) has been proposed as the neural mechanism underlying synaptic plasticity. Moreover, N -methyl- d -aspartate (NMDA) receptors are accepted as the molecular substrate of LTP. It now seems necessary to study the relationship of both LTP and NMDA receptors with the plastic changes taking place, in selected neural structures, during actual learning. Here, we review data on the involvement of the hippocampal CA3–CA1 synapse in the acquisition of classically conditioned eyelid conditioned responses (CRs) in behaving mice. Available data show that LTP, evoked by high-frequency stimulation of Schaffer collaterals, disturbs both the acquisition of CRs and the physiological changes that occur at the CA3–CA1 synapse during learning. Moreover, the administration of NMDA-receptor antagonists is able not only to prevent LTP induction in vivo , but also to hinder the formation of both CRs and functional changes in strength of the CA3–CA1 synapse. Thus, there is experimental evidence relating activity-dependent synaptic changes taking place during actual learning with LTP mechanisms and with the role of NMDA receptors in both processes.