Carbonic anhydrase inhibitors. Inhibition of the β-class enzymes from the fungal pathogens Candida albicans and Cryptococcus neoformans with branched aliphatic/aromatic carboxylates and their derivatives

The inhibition of the β-carbonic anhydrases (CAs, EC 4.2.1.1) from the pathogenic fungi Cryptococcus neoformans (Can2) and Candida albicans (Nce103) with a series of 25 branched aliphatic and aromatic carboxylates has been investigated. Human isoforms hCA I and II were also included in the study for comparison. Aliphatic carboxylates were generally millimolar hCA I and II inhibitors and low micromolar/submicromolar β-CA inhibitors. Aromatic carboxylates were micromolar inhibitors of the four enzymes but some of them showed low nanomolar activity against the fungal pathogenic enzymes. 4-Hydroxy- and 4-methoxy-benzoate inhibited Can2 with K_Is of 9.5-9.9 nM. The methyl esters, hydroxamates, hydrazides and carboxamides of some of these derivatives were also effective inhibitors of the α- and β-CAs investigated here.     

Virtual screening-driven identification of human carbonic anhydrase inhibitors incorporating an original, new pharmacophore

Combinated ligand- and pharmacophore-based virtual screening approaches were used to discover novel potential pharmacophores acting as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors (CAIs). A free database of commercially available compounds was screened through drug-like filters using a four-point pharmacophore, and followed by docking calculation within the active site of an X-ray structure of isoform CA II. One compound, bearing a trifluoro-dihydroxy-propanone moiety, showed an interesting, selective inhibitory activity in low micromolar range against this isoform versus CA I. The chemical originality of this new pharmacophore can represent an important bioisosteric alternative to the sulfonamido-based functionalities, thus leading to the development of a new class of CAIs.     

Six recommendations for improving monitoring of diseases shared with wildlife: examples regarding mycobacterial infections in Spain

Monitoring is needed to identify changes in disease occurrence and to measure the impact of intervention. Using mycobacterial diseases as an example, we discuss herein the pros and cons of the current Spanish Wildlife Disease Surveillance Scheme providing suggestions for monitoring relevant diseases shared with wildlife in other regions facing similar challenges. Six points should be considered. This includes: (1) making sure the disease is properly monitored in the relevant domestic animals or even in humans; (2) also making sure that background information on wildlife population ecology is available to maximize the benefits of the monitoring effort; (3) selecting the appropriate wildlife hosts for monitoring, while being flexible enough to incorporate new ones if research suggests their participation; (4) selecting the appropriate methods for diagnosis and for time and space trend analysis; (5) deciding which parameters to target for monitoring; and finally (6) establishing a reasonable sampling effort and a suitable sampling stratification to ensure detecting changes over time and changes in response to management actions. Wildlife disease monitoring produces knowledge that benefits at least three different agencies, namely, animal health, public health and conservation, and these should combine efforts and resources. Setting up stable, comprehensive and accurate schemes at different spatial scales should become a priority. Resources are always a limiting factor, but experience shows that combined, cross-collaborative efforts allow establishing acceptable schemes with a low enough cost to be sustainable over time. These six steps for monitoring relevant shared diseases can be adapted to many other geographical settings and different disease situations.     

Carbonic anhydrase inhibitors: Synthesis and inhibition of the human carbonic anhydrase isoforms I,II, VII,IX and XII with benzene sulfonamides incorporating 4,5,6,7-tetrabromophthalimide moiety

A series of 4,5,6,7-tetrabromo-1,3-dioxoisoindolin-2-yl benzenesulfonamide derivatives (compounds 1–8) was synthesized by reaction of benzene sulfonamide derivatives with 4,5,6,7-tetrabromophthalic anhydride moiety. These new sulfonamides were investigated as inhibitors of the zinc metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) and more specifically against the human (h) cytosolic isoforms hCA I, II and VII and the transmembrane tumor-associated isoform hCA IX and XII. The new compounds were good hCA I inhibitors (Kis in the range of 143 to >10,000 nM), but were moderately effective, as hCA II inhibitors (Kis of 47–190 nM) and poor hCA VII inhibitors (Kis in the range of 54–175 nM) compared to acetazolamide. The tumor-associated hCA IX was effectively inhibited with Kis ranging between 8.5 and 234 nM and hCA XII with inhibition constants in the range of 6.1–197 nM with high selectivity ratio. The structure–activity relationship (SAR) with this series of sulfonamides is straightforward, with the main features leading to good activity for each isoforms being established. The high sequence hCA alignment homology and molecular docking study of compounds was performed to rationalize the SAR reported over here.     

CaMKII‐dependent phosphorylation of GluK5 mediates plasticity of kainate receptors

Calmodulin‐dependent kinase II (CaMKII) is key for long‐term potentiation of synaptic AMPA receptors. Whether CaMKII is involved in activity‐dependent plasticity of other ionotropic glutamate receptors is unknown. We show that repeated pairing of pre‐ and postsynaptic stimulation at hippocampal mossy fibre synapses induces long‐term depression of kainate receptor (KAR)‐mediated responses, which depends on Ca²⁺ influx, activation of CaMKII, and on the GluK5 subunit of KARs. CaMKII phosphorylation of three residues in the C‐terminal domain of GluK5 subunit markedly increases lateral mobility of KARs, possibly by decreasing the binding of GluK5 to PSD‐95. CaMKII activation also promotes surface expression of KARs at extrasynaptic sites, but concomitantly decreases its synaptic content. Using a molecular replacement strategy, we demonstrate that the direct phosphorylation of GluK5 by CaMKII is necessary for KAR‐LTD. We propose that CaMKII‐dependent phosphorylation of GluK5 is responsible for synaptic depression by untrapping of KARs from the PSD and increased diffusion away from synaptic sites.     

Reproductive biology of the threatened <Emphasis Type="Italic">Lilium pomponium</Emphasis> (Liliaceae), a species endemic to Maritime and Ligurian Alps

Pollination ecology and breeding system of Lilium pomponium L. were studied, and their effect on the reproductive outcome was assessed. This species has high conservation interest in Europe, because it is included in Annex V of the EU Habitat Directive and it is one out of the five Lilium species listed in IUCN Global Red List. To achieve our aim, the pollen vectors as well as the effect of bagging, emasculation and artificial pollination on reproductive output were studied. The most frequent visitor was the Lepidopteran Gonepteryx rhamnii. In general, reproductive outputs were close to zero for all the self-pollination treatments; however, geitonogamy and facilitated selfing seem slightly more efficient than autogamy, as also confirmed by self-compatibility and autofertility indices. Altogether, our results suggest a self-incompatible outcrossing breeding system, with a poor capacity for selfing. Nevertheless, climate change and anthropic threats might promote a shift toward self-fertilization, even maladaptive, favouring the few individuals able to produce selfed seeds.     

Proton pump inhibitors protect mice from acute systemic inflammation and induce long-term cross-tolerance

Incidence of sepsis is increasing, representing a tremendous burden for health-care systems. Death in acute sepsis is attributed to hyperinflammatory responses, but the underlying mechanisms are still unclear. We report here that proton pump inhibitors (PPIs), which block gastric acid secretion, selectively inhibited tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) secretion by Toll-like receptor (TLR)-activated human monocytes in vitro, in the absence of toxic effects. Remarkably, the oversecretion of IL-1β that represents a hallmark of monocytes from patients affected by cryopyrin-associated periodic syndrome is also blocked. Based on these propaedeutic experiments, we tested the effects of high doses of PPIs in vivo in the mouse model of endotoxic shock. Our data show that a single administration of PPI protected mice from death (60% survival versus 5% of untreated mice) and decreased TNF-α and IL-1β systemic production. PPIs were efficacious even when administered after lipopolysaccharide (LPS) injection. PPI-treated mice that survived developed a long-term cross-tolerance, becoming resistant to LPS- and zymosan-induced sepsis. In vitro, their macrophages displayed impaired TNF-α and IL-1β to different TLR ligands. PPIs also prevented sodium thioglycollate-induced peritoneal inflammation, indicating their efficacy also in a non-infectious setting independent of TLR stimulation. Lack of toxicity and therapeutic effectiveness make PPIs promising new drugs against sepsis and other severe inflammatory conditions.Systemic inflammatory response is a critical clinical response to insults of either infectious or non-infectious origin.¹ Severe sepsis and septic shock are more serious clinical forms with a poor outcome.² The incidence of sepsis is continuously increasing;^{1, 2, 3, 4} the mortality rate ranges between 30 and 50% in severe sepsis and septic shock, and patients who survive have a higher risk of mortality compared with the normal population for months and even years.⁵ Although treatment of the underlying infection and circulatory support decrease mortality, sepsis remains a leading cause of death in critically ill patients, and efficacious therapy is missing.⁶Traditionally, the physiopathology of sepsis is attributed to a hyperinflammatory response, the ‘cytokine storm'', that can directly lead to death or favor the insurgence of an immunosuppressive phase during which multiple organ dysfunction occurs.¹ We have recently reproduced in vitro on primary monocytes the cytokine storm: the simultaneous activation of multiple Toll-like receptors (TLRs) results in oxidative stress responsible for a marked enhancement of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) secretion.⁷Proinflammatory cytokines are indeed increased in sepsis. TNF-α⁸ is the first cytokine detected in the serum of septic patients followed by IL-1β.⁹ High-mobility group box chromosomal protein-1 appears well after TNF-α and IL-1β, supporting its role in mortality due to late sepsis.¹⁰The ‘cytokine storm'' theory is presently debated, mainly because clinical trials with cytokine antagonists were unsuccessful.¹¹ Timing of administration and non-optimal association of cytokine-neutralizing agents may be responsible for the failure of clinical trials; however, other factors besides cytokine hypersecretion, including genetic polymorphism in genes for coagulation and fibrinolysis and the insurgence of oxidative stress,² may participate in the genesis of sepsis. A common trait in septic patients is acidosis, which is more pronounced in non-survivors.¹² Acidosis is mainly due to a shift from oxidative phosphorylation to glycolysis, with accumulation of lactate and decrease of pH,¹³ and occurs upstream of the pathologic events (release of toxic substances, vasoconstriction, endothelial damage) that lead to cell and patient death.¹² Acidosis is also a feature of inflammatory microenvironments:^{14, 15} upon activation, inflammatory cell metabolism shifts toward aerobic glycolysis,¹⁶ with consequent decrease of extracellular pH. Extracellular acidosis is proinflammatory: it induces inflammatory genes^{14, 15} and increases processing and secretion of IL-1β¹⁷ in a NOD-like receptor (NLR) family, pyrin domain containing 3 (NLRP3) inflammasome-dependent^{18, 19} or -independent²⁰ manner.Proton pump inhibitors (PPIs) are a family of prodrugs that, activated by low pH,²¹ are highly efficacious in reducing acidic secretion by gastric cells and therefore largely used in the treatment of peptic ulcers and reflux esophagitis.²² PPIs are also effective against tumors, which are similarly characterized by low pH.^{23, 24} Furthermore, PPIs have been found to exert anti-inflammatory effects unrelated to the inhibition of gastric acid production, although the underlying mechanisms remain to be elucidated.²⁵Here we show that, in vitro, PPIs inhibit the production of proinflammatory cytokines by monocytes stimulated with TLR agonists; in vivo, in a murine model of lethal endotoxic shock,^{8, 26} PPIs protect against lipopolysaccharide (LPS)-induced mortality. Interestingly, mice cured by PPIs develop cross-tolerance: not only are they resistant to a second challenge with LPS but also respond better to zymosan injection.