The indole motif is essential for the antitrypanosomal activity of N5-substituted paullones.

Severe infections with potentially fatal outcomes are caused by parasites from the genera Trypanosoma and Leishmania (class Kinetoplastea). The diseases affect people of remote areas in the tropics and subtropics with limited access to adequate health care. Besides insufficient diagnostics, treatment options are limited, with tenuous developments in recent years. Therefore, new antitrypanosomal antiinfectives are required to fight these maladies. In the presented approach, new compounds were developed and tested on the target trypanothione synthetase (TryS). This enzyme is crucial to the kinetoplastids' unique trypanothione-based thiol redox metabolism and thus for pathogen survival. Preceding studies have shown that N5-substituted paullones display antitrypanosomal activity as well as TryS inhibition. Herein, this compound class was further examined regarding the structure-activity relationships (SAR). Diverse benzazepinone derivatives were designed and tested in cell-based assays on bloodstream Trypanosoma brucei brucei (T. b. brucei) and intracellular amastigotes of Leishmania infantum (L. infantum) as well as in enzyme-based assays on L. infantum TryS (LiTryS) and T. b. brucei TryS (TbTryS). While an exchange of just the substituent in the 9-position of paullones led to potent inhibitors on LiTryS and T. b. brucei parasites, new compounds lacking the indole moiety showed a total loss of activity in both assays. Conclusively, the indole as part of the paullone structure is pivotal for keeping the TryS inhibitory and antitrypanosomal activity of this substance class.

Discovery of Antitrypanosomal Indolylacetamides by a Deconstruction-Optimization Strategy Applied to Paullones.

The parasitic kinetoplastid diseases Leishmaniasis, Chagas disease and Human African Trypanosomiasis constitute serious threats for populations throughout the (sub-)tropics. Most available drugs to treat these diseases possess inadequate properties and candidates to fill the drug pipeline are urgently needed. Paullone-N5 -acetamides inhibit trypanothione synthetase (TryS), an essential kinetoplastid enzyme, and exhibit antiparasitic activity in the low micromolar range, but lack the desired selectivity against mammalian cells (selectivity index (SI):<10). With the aim to identify the paullones' moieties responsible for TryS inhibition and bioactivity, we applied molecular simplification and ring disconnection approaches. The new indolylacetamides lost activity against the expected molecular target (TryS) compared to the reference paullone MOL2008 (Leishmania infantum TryS IC50 : 150 nM; Trypanosoma brucei bloodstream form EC50 : 4.3 µM and SI: 2.4). However, several of them retained potency (T. b. brucei EC50 : 2.4-12.0 µM) and improved selectivity (SI: 5 to >25).

Mechanistic and biological characterisation of novel N5-substituted paullones targeting the biosynthesis of trypanothione in Leishmania.

Trypanothione synthetase (TryS) produces N1,N8-bis(glutathionyl)spermidine (or trypanothione) at the expense of ATP. Trypanothione is a metabolite unique and essential for survival and drug-resistance of trypanosomatid parasites. In this study, we report the mechanistic and biological characterisation of optimised N5-substituted paullone analogues with anti-TryS activity. Several of the new derivatives retained submicromolar IC50 against leishmanial TryS. The binding mode to TryS of the most potent paullones has been revealed by means of kinetic, biophysical and molecular modelling approaches. A subset of analogues showed an improved potency (EC50 0.5-10 µM) and selectivity (20-35) against the clinically relevant stage of Leishmania braziliensis (mucocutaneous leishmaniasis) and L. infantum (visceral leishmaniasis). For a selected derivative, the mode of action involved intracellular depletion of trypanothione. Our findings shed light on the molecular interaction of TryS with rationally designed inhibitors and disclose a new set of compounds with on-target activity against different Leishmania species.

UK undergraduate aspirations and attitudes survey: do we have a perception problem in clinical radiology?

AIM: To understand medical students' potential long-term career choices, with particular reference to radiology, and their current perceptions and experiences of radiology and radiologists, with the aim to help inform future initiatives for undergraduate and early postgraduate medical education and workforce strategies. MATERIALS AND METHODS: An invitation to an online survey was sent to all undergraduate medical students at a large UK medical school, using Likert-style, multiple choice and ranking questions. A quantitative approach was used to explore these responses (n=328). RESULTS: Radiology ranked only 10th out of 14 specialties for long-term career preference amongst medical student respondents (n=328). Radiology was judged as being "low status", but enabled a "good" work-life balance. Medical students considered making an impact on patient diagnosis and level of intellectual challenge as positive influences of a potential career in radiology. Perception of radiology by the public, patient relationship/contact time, perception of radiology by other clinicians, variation of work, and radiology work environment were all perceived more negatively. CONCLUSION: Radiology remains a specialty with limited exposure and experience for undergraduate students, who appear to be incompletely aware of the scope and range of the work of modern radiologists. Greater exposure to radiology teaching in the undergraduate curriculum and placements in radiology departments may increase and expedite medical students' understanding and enjoyment of radiology at a juncture where demand for radiology services is increasing rapidly in the UK.

Trypanothione synthetase confers growth, survival advantage and resistance to anti-protozoal drugs in Trypanosoma cruzi.

BACKGROUND: Chagas cardiomyopathy, caused by Trypanosoma cruzi infection, continues to be a neglected illness, and has a major impact on global health. The parasite undergoes several stages of morphological and biochemical changes during its life cycle, and utilizes an elaborated antioxidant network to overcome the oxidants barrier and establish infection in vector and mammalian hosts. Trypanothione synthetase (TryS) catalyzes the biosynthesis of glutathione-spermidine adduct trypanothione (T(SH)2) that is the principal intracellular thiol-redox metabolite in trypanosomatids. METHODS AND RESULTS: We utilized genetic overexpression (TryShi) and pharmacological inhibition approaches to examine the role of TryS in T. cruzi proliferation, tolerance to oxidative stress and resistance to anti-protozoal drugs. Our data showed the expression and activity of TryS was increased in all morphological stages of TryShi (vs. control) parasites. In comparison to controls, the TryShi epimastigotes (insect stage) recorded shorter doubling time, and both epimastigotes and infective trypomastigotes of TryShi exhibited 36-71% higher resistance to H2O2 (50-1000 µM) and heavy metal (1-500 µM) toxicity. Treatment with TryS inhibitors (5-30 µM) abolished the proliferation and survival advantages against H2O2 pressure in a dose-dependent manner in both TryShi and control parasites. Further, epimastigote and trypomastigote forms of TryShi (vs. control) T. cruzi tolerated higher doses of benznidazole and nifurtimox, the drugs currently administered for acute Chagas disease treatment. CONCLUSIONS: TryS is essential for proliferation and survival of T. cruzi under normal and oxidant stress conditions, and provides an advantage to the parasite to develop resistance against currently used anti-trypanosomal drugs. TryS indispensability has been chemically validated with inhibitors that may be useful for drug combination therapy against Chagas disease.

5-Substituted 3-chlorokenpaullone derivatives are potent inhibitors of Trypanosoma brucei bloodstream forms.

Trypanothione synthetase is an essential enzyme for kinetoplastid parasites which cause highly disabling and fatal diseases in humans and animals. Inspired by the observation that N(5)-substituted paullones inhibit the trypanothione synthetase from the related parasite Leishmania infantum, we designed and synthesized a series of new derivatives. Although none of the new compounds displayed strong inhibition of Trypanosoma brucei trypanothione synthetase, several of them caused a remarkable growth inhibition of cultivated Trypanosoma brucei bloodstream forms. The most potent congener 3a showed antitrypanosomal activity in double digit nanomolar concentrations and a selectivity index of three orders of magnitude versus murine macrophage cells.

High-speed cannula detachment into the eye during hydrodissection.

Detachment of a hydrodissection cannula during a phacoemulsification procedure appeared to produce no adverse sequelae during surgery. Day 1 postoperatively, two nonpenetrating hemorrhagic retinal lesions were identified; there was no evidence of posterior capsular perforation. Day 6 postoperatively, the pupil was temporally peaked by a fine vitreous strand running to the main-port incision in the superotemporal cornea. This was divided with Nd:YAG laser, and argon laser was applied to encircle the two retinal lesions. Postoperative uncorrected visual acuity remained 6/4 at day 1, day 6, and week 4 (3 weeks after laser application) follow-up visits. Surgeons must accept responsibility for confirming the integrity of the cannula and syringe connection before beginning hydrodissection, which can be highly destructive to intracameral structures.

Acute intraoperative rock-hard eye syndrome and its management.

PURPOSE: To evaluate the use of pars plana needle aspiration of retrolenticular fluid in the immediate management of an acute intraoperative rock-hard eye syndrome (AIRES). SETTING: Private practice, Sydney, Australia. DESIGN: Retrospective case series. METHODS: Data over an 18-month period were collected to evaluate efficacy, complications, and visual outcomes in patients who had pars plana needle aspiration for management of AIRES, which is an acute intraoperative shallowing of the anterior chamber and a marked increase in intraocular pressure (IOP) during phacoemulsification cataract surgery but without evidence of a choroidal hemorrhage. Preoperative and postoperative (1 day, 1 week, and 1 month) data were evaluated. Resolution of AIRES and postoperative posterior segment status, IOP, and corrected distance visual acuity (CDVA) were the main outcome measures. RESULTS: Acute intraoperative rock-hard eye syndrome occurred in 6 (1.45%) of 413 surgeries. All 6 patients were women with a mean age of 81 years. Four patients had dense nuclear cataracts. In each case, the anterior chamber depth and IOP normalized immediately after pars plana needle aspiration and the procedure concluded uneventfully. Mild vitreous hemorrhage was observed in 1 patient postoperatively. At 1 month, the IOP was normal in 5 of 6 cases, while the CDVA was 20/12 in 5 of 6 cases. CONCLUSION: Although the etiology of AIRES is iatrogenic, immediate resolution was achieved uneventfully with pars plana needle aspiration, which appears to be a safe management technique with satisfactory outcomes.

P2Y2 receptor agonists for the treatment of dry eye disease: a review.

Recent advances in the understanding of dry eye disease (DED) have revealed previously unexplored targets for drug therapy. One of these drugs is diquafosol, a uridine nucleotide analog that is an agonist of the P2Y2 receptor. Several randomized controlled trials have demonstrated that the application of topical diquafosol significantly improves objective markers of DED such as corneal and conjunctival fluorescein staining and, in some studies, tear film break-up time and Schirmer test scores. However, this has been accompanied by only partial improvement in patient symptoms. Although evidence from the literature is still relatively limited, early studies have suggested that diquafosol has a role in the management of DED. Additional studies would be helpful to delineate how different subgroups of DED respond to diquafosol. The therapeutic combination of diquafosol with other topical agents also warrants further investigation.

Successful "medical" orbital decompression with adjunctive rituximab for severe visual loss in IgG4-related orbital inflammatory disease with orbital myositis.

IgG4-related disease (IgG4-RD) is an inflammatory condition of unknown etiology that can cause tumefactive lesions in a number of tissues and organs, including the orbit and ocular adnexa. Diagnostic criteria for IgG4-RD, including pathology and clinical features and pathology, have been recently proposed. This study presents the first case of unilateral acute visual loss secondary to IgG4-related orbital inflammatory disease with orbital myositis that was complicated by severe compressive optic neuropathy. After initial treatment with pulsed intravenous methylprednisolone, followed by rituximab and radiotherapy, there was a marked improvement in orbital inflammation and clinical and radiological improvement in the compressive optic neuropathy. After 9 months of follow up, the orbital inflammatory disease remained in remission.

AMPAKINE enhancement of social interaction in the BTBR mouse model of autism.

Autism is a neurodevelopmental disorder in which the first diagnostic symptom is unusual reciprocal social interactions. Approximately half of the children diagnosed with an autism spectrum disorder also have intellectual impairments. General cognitive abilities may be fundamental to many aspects of social cognition. Cognitive enhancers could conceivably be of significant benefit to children and adults with autism. AMPAKINE compounds are a novel class of pharmacological agents that act as positive modulators of AMPA receptors to enhance excitatory glutamatergic neurotransmission. This class of compounds was reported to improve learning and memory in several rodent and non-human primate tasks, and to normalize respiratory abnormalities in a mouse model of Rett syndrome. Here we evaluate the actions of AMPA compounds in adult male and female BTBR mice, a well characterized mouse model of autism. Acute treatment with CX1837 and CX1739 reversed the deficit in sociability in BTBR mice on the most sensitive parameter, time spent sniffing a novel mouse as compared to time spent sniffing a novel object. The less sensitive parameter, time in the chamber containing the novel mouse versus time in the chamber containing the novel object, was not rescued by CX1837 or CX1739 treatment. Preliminary data with CX546, in which ß-cyclodextrin was the vehicle, revealed behavioral effects of the acute intraperitoneal and oral administration of vehicle alone. To circumvent the artifacts introduced by the vehicle administration, we employed a novel treatment regimen using pellets of peanut butter for drug delivery. Absence of vehicle treatment effects when CX1837 and CX1739 were given in the peanut butter pellets, to multiple cohorts of BTBR and B6 control mice, confirmed that the pharmacologically-induced improvements in sociability in BTBR were not confounded by the administration procedures. The highest dose of CX1837 improved the cognitive deficit in novel object recognition in BTBR. No drug effects were detected on the high levels of repetitive self-grooming in BTBR. In open field tests, CX1837 and CX1739 did not induce hyperactivity or sedation in either strain. It is interesting to speculate that the ability of CX1837 and CX1739 to restore aspects of sociability in BTBR mice could utilize synaptic mechanisms regulating social cognition, suggesting a potential pharmacological target for interventions to treat symptoms of autism. This article is part of a Special Issue entitled 'Cognitive Enhancers'.

Engineering the substrate specificity of Bacillus megaterium cytochrome P-450 BM3: hydroxylation of alkyl trimethylammonium compounds.

Oligonucleotide-directed mutagenesis has been used to replace arginine-47 with glutamate in cytochrome P-450 BM3 from Bacillus megaterium and in its haem domain. The mutant has been characterized by sequencing, mass spectrometry, steady-state kinetics and by optical and NMR measurements of substrate binding. The mutant retains significant catalytic activity towards C12-C16 fatty acids, catalysing hydroxylation in the same (omega-1, omega-2, omega-3) positions with kcat/Km values a factor of 14-21 lower. C12-C16 alkyl trimethylammonium compounds are relatively poor substrates for the wild-type enzyme, but are efficiently hydroxylated by the arginine-47-->glutamate mutant at the omega-1, omega-2 and omega-3 positions, with kcat values of up to 19 s-1. Optical spectroscopy shows that the binding of the C14 and C16 alkyl trimethylammonium compounds to the mutant is similar to that of the corresponding fatty acids to the wild-type enzyme. Paramagnetic relaxation measurements show that laurate binds to the ferric state of the mutant in a significantly different position, 1.5 A closer to the iron, than seen in the wild-type, although this difference is much smaller ( approximately 0.2 A) in the ferrous state of the complex. The binding of a substrate having the same charge as residue 47 to the ferric state of the enzyme is roughly ten times weaker than that of a substrate having the opposite charge (and thus is able to make an ion-pair interaction with this residue). The results are discussed in the light of the three-dimensional structure of the enzyme.

A single mutation in cytochrome P450 BM3 changes substrate orientation in a catalytic intermediate and the regiospecificity of hydroxylation.

Phenylalanine 87 of Bacillus megaterium cytochrome P450 BM3, a residue close to the heme in the substrate binding pocket, has been replaced by alanine by site-directed mutagenesis. The substitution had no effect on the rate of hydroxylation of laurate and increased the affinity for laurate of both the intact enzyme and its heme domain by 2.6-6-fold in the ferric state. NMR paramagnetic relaxation measurements showed that in the initial ferric enzyme-substrate complex, where the substrate binds relatively far from the heme, the substitution had no effect on the position or orientation of the bound substrate. However, in the next intermediate in the catalytic cycle, the reduced enzyme, the position of the bound substrate was altered so that the terminal methyl group was 3.1 A from the iron in the mutant, compared to 5.1 A in the wild-type enzyme. Analysis of the products of the action of the enzyme on laurate and myristate showed that the mutant catalyzed hydroxylation almost exclusively at the omega position, in marked contrast to the wild-type enzyme, with which no hydroxylation at this position was observed.