Bioisosteric ferrocenyl aminoquinoline-benzimidazole hybrids: Antimicrobial evaluation and mechanistic insights.

Phenyl- and bioisosteric ferrocenyl-derived aminoquinoline-benzimidazole hybrid compounds were synthesised and evaluated for their in vitro antiplasmodial activity against the chloroquine-sensitive NF54 and multi-drug resistant K1 strains of the human malaria parasite, Plasmodium falciparum. All compounds were active against the two strains, generally showing enhanced activity in the K1 strain, with resistance indices less than 1. Cytotoxicity studies using Chinese hamster ovarian cells revealed that the hybrids were relatively non-cytotoxic and demonstrated selective killing of the parasite. Based on favourable in vitro antiplasmodial and cytotoxicity data, the most active phenyl (4c) and ferrocenyl (5b) hybrids were tested in vivo against the rodent Plasmodium berghei mouse model. Both compounds caused a reduction in parasitemia relative to the control, with 5c displaying superior activity (92% reduction in parasitemia at 4 × 50 mg/kg oral doses). The most active phenyl and ferrocenyl derivatives showed inhibition of ß-haematin formation in a NP-40 detergent-mediated assay, indicating a possible contributing mechanism of antiplasmodial action. The most active ferrocenyl hybrid did not display appreciable reactive oxygen species (ROS) generation in a ROS-induced DNA cleavage gel electrophoresis study. The compounds were also screened for their in vitro activity against Mycobacterium tuberculosis. The hybrids containing a more hydrophobic substituent had enhanced activity (<32.7 µM) compared to those with a less hydrophobic substituent (>62.5 µM).

The bacterial redox signaller pyocyanin as an antiplasmodial agent: comparisons with its thioanalog methylene blue.

The quorum sensor and signalling molecule pyocyanin (PYO) contributes significantly to the pathophysiology of Pseudomonas aeruginosa infections. Comparison to phenothiazine drugs suggests that the antimalarial compound methylene blue (MB) can be regarded as a sulfur analog of PYO. This working hypothesis would explain why the synthetic drug MB behaves as a compound shaped in biological evolution. Here we report on redox-associated biological and biochemical properties of PYO in direct comparison to its synthetic analog MB. We quantitatively describe the reactivity of both compounds toward cellular reductants, the reactivity of their reduced leuco-forms towards O2, and their interactions with FAD-containing disulfide reductases. Furthermore, the interaction of PYO with human glutathione reductase was studied in structural detail by x-ray crystallography, showing that a single PYO molecule binds to the intersubunit cavity of the enzyme. Like MB, also PYO was also found to be active against blood schizonts of the malaria parasite P. falciparum in vitro. Furthermore, both compounds were active against the disease transmitting gametocyte forms of the parasites, which was systematically studied in vitro. As shown for mice, PYO is too toxic to be used as a drug. It may, however, have antimalarial activity in numerous human patients with concomitant Pseudomonas infections. MB, in contrast to PYO, is well tolerated and represents a promising agent for MB-based combination therapies against malaria. Current and future clinical studies can be guided by the comparisons between MB and PYO reported here. Additionally, it is of interest to study if and to what extent the protection from malaria in patients with cystic fibrosis or with severe wound infections is based on PYO produced by Pseudomonas species.

Screening medicinal plants for the detection of novel antimalarial products applying the inhibition of ß-hematin formation.

The identification of novel scaffolds for the development of effective and safe treatments to fight malaria is urgently needed. One of the main opportunities is the discovery of new molecules from natural origin. A simple, robust and cost-effective colorimetric assay based on the inhibition of ß-hematin has been adapted to routinely screen plant extracts with the ultimate goal to identify novel antimalarial ingredients. The development of this assay has included a careful optimization of all critical experimental parameters. The ß-hematin assay can be completed in less than one working day, requiring a 96-well UV-vis plate reader and low-cost commercially available reagents using a standard operating protocol. It can be used on its own or in combination with the well-known Plasmodium growth inhibition assay and has the obvious merit to be informative at the early stage of drug discovery regarding the mechanism of action of the actives. A total of 40 diverse natural products and 219 plants extracts were tested. Good correlations in respect with specificity (pure compounds 85%, extracts 93%) and positive predictive value (pure compounds 72%, extracts 50%) were obtained in comparison with Plasmodium growth inhibition assay that was used as the reference assay.

Plasmodium berghei ANKA: selection of resistance to piperaquine and lumefantrine in a mouse model.

We have selected piperaquine (PQ) and lumefantrine (LM) resistant Plasmodium berghei ANKA parasite lines in mice by drug pressure. Effective doses that reduce parasitaemia by 90% (ED(90)) of PQ and LM against the parent line were 3.52 and 3.93 mg/kg, respectively. After drug pressure (more than 27 passages), the selected parasite lines had PQ and LM resistance indexes (I(90)) [ED(90) of resistant line/ED(90) of parent line] of 68.86 and 63.55, respectively. After growing them in the absence of drug for 10 passages and cryo-preserving them at -80 degrees C for at least 2 months, the resistance phenotypes remained stable. Cross-resistance studies showed that the PQ-resistant line was highly resistant to LM, while the LM-resistant line remained sensitive to PQ. Thus, if the mechanism of resistance is similar in P. berghei and Plasmodium falciparum, the use of LM (as part of Coartem) should not select for PQ resistance.

SOCS1 deficiency results in accelerated mammary gland development and rescues lactation in prolactin receptor-deficient mice.

Prolactin is essential for proliferation and differentiation of the developing mammary gland. We have explored a role for Suppressor of Cytokine Signaling 1 (SOCS1) as a modulator of the prolactin response using mice deficient in SOCS1, which were rescued from neonatal death by deletion of the Interferon gamma (IFN gamma) gene. SOCS1(-/-)/IFN gamma(-/-) mice exhibited accelerated lobuloalveolar development in the mammary gland during late pregnancy and precocious lactation. Significantly, the lactogenic defect in prolactin receptor heterozygous females could be rescued by deletion of a single SOCS1 allele. These findings establish a role for SOCS1 as a negative regulator of prolactin signaling and suggest that SOCS1 is required for the prevention of lactation prior to parturition.

Mechanisms and kinetics of procathepsin D activation.

In vitro, procathepsin D is activated to pseudocathepsin D by incubation at low pH. To investigate the mechanism of this activation, recombinant human procathepsin D and two mutants were generated in a baculovirus expression system. One mutant carried a point mutation within the catalytic domain, which resulted in a catalytically inactive enzyme form (D77A). The other carried a point mutation within the propeptide, which prevented activation by processing at the 'autoproteolysis-site' (L26P). Neither mutant is capable of processing itself to form pseudocathepsin D, and L26P is not able to process D77A. Despite the inability of L26P to cleave either its own or a wild-type prosequence, it did exhibit activity against a synthetic peptide substrate. The ability of intact precursor (zymogen) to cleave a peptide, but not a protein substrate, offers new insights into the mechanism of inhibition by the propeptide. Mature cathepsin D can process the inactive D77A mutant to the pseudoform, demonstrating that processed species are capable of cleaving zymogen molecules in an intermolecular interaction. In addition, kinetic studies provide evidence for a two-phase mechanism for the conversion of procathepsin D to pseudocathepsin D, one phase where the first molecules of pseudocathepsin D are formed at a low rate and a second phase where the process is autocatalytically accelerated by newly formed pseudocathepsin D molecules. Finally, with the help of the mutants L26P and D77A it was observed that at least two additional proteinase activities, found in conditioned media from insect cell culture, are capable of activating procathepsin D by cleaving it within the proregion. This observation suggests that there are likely to be multiple proteinases in the extracellular matrix that are capable of activating procathepsin D, thereby triggering the second autocatalytic phase. This may also be important for solid tumors, where the presence of cathepsin D has been correlated with tumor growth and invasion.

One-step purification of cathepsin D by affinity chromatography using immobilized propeptide sequences.

In vivo, active cathepsin D proteinase is generated by removal of a 44-residue propeptide at its N-terminus. Here we report that mature cathepsin D and pseudocathepsin D (a partially activated form of cathepsin D with 25 amino acid residues removed from the propeptide) bind to the immobilized propeptide, while procathepsin D does not. The N-terminal 25 amino acid residues of the propeptide are sufficient for this binding. Based on this observation, a simple one-step procedure was developed to purify mature cathepsin D from whole cell extracts to near homogeneity. This method has the advantage over existing affinity-purification systems that active forms of the proteinase can be separated from inactive precursors and other aspartic proteinases. Furthermore, this technique was effective for pepsin as well, suggesting it may have general utility for all activated aspartic proteinases and perhaps other families of proteinases.

Tetracaine, propranolol and trifluoperazine inhibit thyrotropin releasing hormone-induced prolactin secretion from GH3 cells by displacing membrane calcium: further evidence that TRH acts to mobilize cellular calcium.

TRH stimulation of prolactin release from GH3 cells is associated with loss of cellular Ca2+. Chlortetracycline (CTC), a fluorescent probe of Ca2+ in biological membranes, was previously employed to monitor indirectly changes in membrane Ca2+ in GH3 cells. Tetracaine, propranolol and trifluoperazine, agents that are known to displace Ca2+ from biological membranes, were utilized to demonstrate more rigorously that TRH affects cellular membrane Ca2+ in GH3 cells. Tetracaine (1 mM), propranolol (1 mM), and trifluoperazine (0.03 mM) inhibited basal and TRH-stimulated prolactin release, decreased cellular 45Ca2+ content and decreased cell-associated CTC fluorescence. Most importantly, these agents abolished the decrease in CTC fluorescence induced by TRH. These data suggest that tetracaine, propranolol and trifluoperazine displace membrane Ca2+ in intact GH3 cells and offer further evidence that TRH acts to mobilize cellular Ca2+ from a membrane-bound pool(s) during stimulation of GH3 cells.