Anti-infectives: can cellular screening deliver?

In an era of emerging and reemerging infectious diseases, and increasing multidrug resistance, the need to identify novel therapy is imperative. Unfortunately, the recent shift of the drug discovery paradigm from cellular screening to target-based approaches has not delivered the anticipated benefits. A recent renaissance of the traditional cell-based approach, on the other hand, has yielded several clinical candidates. Three successful examples are illustrated in this review, namely spiroindolone, thiazolidinone, and diarylquinoline for the treatment of malaria, hepatitis C virus, and tuberculosis, respectively. We describe in detail their identification, mechanism of action (MoA), and common features in the chemical structures. The challenges of the cell-based approach for anti-infective drug discovery are also discussed. We propose a shift from standard libraries to synthetic natural-product-like compound collections to improve the success of phenotypic lead finding and to facilitate the validation of hits.     

High throughput screening methods for ω-transaminases

Recently, ω-transaminases have been increasingly used to synthesize amine compounds by reductive amination of prochiral ketones which are of high pharmacological significance. However, the conventional methods for evaluating these enzymes are time consuming and have often been regarded as a bottle neck in developing these enzymes as industrial biocatalysts. In the past few years, several high throughput screening methods have been developed for fast evaluation and identification of ω-transaminase. This review summarizes the various methodologies developed for rapidly screening ω-transaminases.     

How good is your screening library?

Efficient library design is an ongoing challenge for investigators seeking novel ligands for proteins, whether for drug discovery or chemical biology. Strategies that add neglected chemistry or exclude unproductive compounds are two dominant recent themes, as is a growing awareness of molecular complexity and its implications. The choice of how complex molecules in screening libraries should be often amounts to how big they should be. Small, simple molecules have lower affinities and must be screened at high concentration, but they will also have higher hit rates. Larger compounds, on the other hand, will often more closely resemble final drugs, but because they are more highly functionalized and specific, they will have much lower hit rates. The best general-purpose screening libraries may well be those of intermediate complexity that are free of artifact-causing nuisance compounds.     

Novel screening methods — biosensors

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Nutritionele screening bij de oncogeriatrische patiënt

Geriatric patients are not defined by their age but by their general profile. Ageing is characterized by loss of organ function together with a reduced capability for adapting to changes in the environment (loss of homeostatic mechanisms) leading to frailty. In the older patient with cancer, there can be problems of dietary intake next to the effects of ageing per se. On top of this situation, the deleterious effects of the inflammatory processes induced by the tumour are superimposed. When these changes are translated into nutritional concepts, it is clear that, in the older cancer patient, there is a strong overlap of starvation, sarcopenia, and cachexia. Nutritional assessment should be part of the routine preliminary evaluation of the older oncology patient. Difference should be made between assessment of risk and actual nutritional status, which should be assessed with specific malnutrition indices. Body weight assessment with specific attention to unintended weight loss is essential in this evaluation. One should recognise the fact that body mass index (BMI) should be interpreted with caution, but that a low value for BMI still heralds an increased malnutrition risk. This increased alertness for nutritional problems has a lot to offer in the willingness for early intervention. The nutritional assessment, however, must be framed in a larger comprehensive geriatric assessment addressing several functional domains.     

Performance of the CareStart™ G6PD deficiency screening test, a point-of-care diagnostic for primaquine therapy screening

Development of reliable, easy-to-use, rapid diagnostic tests (RDTs) to detect glucose-6-phosphate dehydrogenase (G6PD) deficiency at point of care is essential to deploying primaquine therapies as part of malaria elimination strategies. We assessed a kit under research and development called CareStart? G6PD deficiency screening test (Access Bio, New Jersey, USA) by comparing its performance to quantitative G6PD enzyme activity using a standardized spectrophotometric method ('gold standard'). Blood samples (n?=?903) were collected from Cambodian adults living in Pailin province, western Cambodia. G6PD enzyme activities ranged from 0 to 20.5 U/g Hb (median 12.0 U/g Hg). Based on a normal haemoglobin concentration and wild-type G6PD gene, the normal values of G6PD enzymatic activity for this population was 3.6 to 20.5 U/g Hg (95(th) percentiles from 5.5 to 17.2 U/g Hg). Ninety-seven subjects (10.7%) had <3.6 U/g Hg and were classified as G6PD deficient. Prevalence of deficiency was 15.0% (64/425) among men and 6.9% (33/478) among women. Genotype was analyzed in 66 G6PD-deficient subjects and 63 of these exhibited findings consistent with Viangchang genotype. The sensitivity and specificity of the CareStart? G6PD deficiency screening test was 0.68 and 1.0, respectively. Its detection threshold was <2.7 U/g Hg, well within the range of moderate and severe enzyme deficiencies. Thirteen subjects (1.4%, 12 males and 1 female) with G6PD enzyme activities <2 U/g Hg were falsely classified as "normal" by RDT. This experimental RDT test here evaluated outside of the laboratory for the first time shows real promise, but safe application of it will require lower rates of falsely "normal" results.     

A new resorufin-based α-glucosidase assay for high-throughput screening

Mutations in α-glucosidase cause accumulation of glycogen in lysosomes, resulting in Pompe disease, a lysosomal storage disorder. Small molecule chaperones that bind to enzyme proteins and correct the misfolding and mistrafficking of mutant proteins have emerged as a new therapeutic approach for the lysosomal storage disorders. In addition, α-glucosidase is a therapeutic target for type II diabetes, and α-glucosidase inhibitors have been used in the clinic as alternative treatments for this disease. We have developed a new fluorogenic substrate for the α-glucosidase enzyme assay, resorufin α-d-glucopyranoside. The enzyme reaction product of this new substrate emits at a peak of 590 nm, reducing the interference from fluorescent compounds seen with the existing fluorogenic substrate, 4-methylumbelliferyl-α-d-glucopyranoside. Also, the enzyme kinetic assay can be carried out continuously without the addition of stop solution due to the lower pK_a of the product of this substrate. Therefore, this new fluorogenic substrate is a useful tool for the α-glucosidase enzyme assay and will facilitate compound screening for the development of new therapies for Pompe disease.     

A chromogenic cephalosporin for β-lactamase inhibitor screening assays

Production of β-lactamases is the primary mechanism of antibiotic resistance employed by gram-negative pathogens. Chromogenic β-lactams are important reagents for detection and assay of β-lactamases, but limited commercial availability and exorbitant pricing of these compounds are prohibitive. Here we describe a straightforward synthesis of a chromogenic cephalosporin for β-lactamase assay that gives an overall yield of 74%. On hydrolysis, its λ(max) undergoes a bathochromic shift that is easy to see and measure spectrophotometrically with a Δε(442 nm) of 14,500cm(-1)M(-1). This compound was shown to be a substrate for a variety of β-lactamases.     

Phenotypic screening meets natural products in drug discovery†

The Nobel Prize in Physiology or Medicine 2015 was awarded for discoveries related to the control of parasitic diseases using natural products of microbial and plant origin. In current drug discovery programs, synthesized compounds are widely used as a screening source; however, this award reminds us of the importance of natural products. Here, we introduce our phenotypic screening methods based on changes in cell morphology and discuss their effectiveness and impact for natural products in drug discovery.     

How should we interpret noncompliance with screening mammography?

Primary care practitioners have an important role to play in recommending breast cancer screening to patients in the target age group. In this issue of CMAJ (see pages 1335 to 1343) Dr. Marie-Dominique Beaulieu and associates report the results of a program designed to maximize utilization of screening mammography. Only two thirds of eligible women for whom screening mammography was prescribed obtained a mammogram within the 2-month study period. However, when taken in context, this compliance rate is fairly encouraging. There are many possible reasons for noncompliance such as a need for more information or for repeated suggestions. Family physicians should not become disheartened in their efforts to increase the use of screening procedures and may find that collaboration with others in giving consistent messages will help to maximize screening rates within their patient population.     

E-pharmacophore-based virtual screening to identify GSK-3β inhibitors

Glycogen synthase kinase-3β (GSK-3β) is a serine/threonine kinase which has attracted significant attention during recent years in drug design studies. The deregulation of GSK-3β increased the loss of hippocampal neurons by triggering apoptosis-mediating production of neurofibrillary tangles and alleviates memory deficits in Alzheimer’s disease (AD). Given its role in the formation of neurofibrillary tangles leading to AD, it has been a major therapeutic target for intervention in AD, hence was targeted in the present study. Twenty crystal structures were refined to generate pharmacophore models based on energy involvement in binding co-crystal ligands. Four common e-pharmacophore models were optimized from the 20 pharmacophore models. Shape-based screening of four e-pharmacophore models against nine established small molecule databases using Phase v3.9 had resulted in 1800 compounds having similar pharmacophore features. Rigid receptor docking (RRD) was performed for 1800 compounds and 20 co-crystal ligands with GSK-3β to generate dock complexes. Interactions of the best scoring lead obtained through RRD were further studied with quantum polarized ligand docking (QPLD), induced fit docking (IFD) and molecular mechanics/generalized Born surface area. Comparing the obtained leads to 20 co-crystal ligands resulted in 18 leads among them, lead1 had the lowest docking score, lower binding free energy and better binding orientation toward GSK-3β. The 50?ns MD simulations run confirmed the stable nature of GSK-3β-lead1 docking complex. The results from RRD, QPLD, IFD and MD simulations confirmed that lead1 might be used as a potent antagonist for GSK-3β.     

Is population screening for abdominal aortic aneurysm cost-effective?

Background

The adverse effects of tobacco abuse on cardiovascular outcomes are well-known. However, the impact of passive smoke exposure on angina status and therapeutic response is less well-established. We examined the impact of second-hand smoke (SHS) exposure on symptomatic improvement in patients with chronic ischemic coronary disease undergoing enhanced external counterpulsation (EECP).

Methods

This observational study included 1,026 non-smokers (108 exposed and 918 not-exposed to SHS) from the Second International EECP Patient Registry. We also assessed angina response in 363 current smokers. Patient demographics, symptomatic improvement and quality of life assessment were determined by self-report prior and after EECP treatment.

Results

Non-smoking SHS subjects had a lower prevalence of prior revascularization (85% vs 90%), and had an increased prevalence of stroke (13% vs 7%) and prior smoking (72% vs 61%; all p < 0.05) compared to non-smokers without SHS exposure. Despite comparable degrees of coronary disease, baseline angina class, medical regimens and side effects during EECP, fewer SHS non-smokers completed a full 35-hour treatment course (77% vs 85%, p = 0.020) compared to non-smokers without SHS. Compared to non-smokers without SHS, non-smoking SHS subjects had less angina relief after EECP (angina class decreased ≥ 1 class: 68% vs 79%; p = 0.0082), both higher than that achieved in current smokers (66%). By multivariable logistic regression, SHS exposure was an independent predictor of failure to symptomatic improvement after EECP among non-smokers (OR 1.81, 95% confidence intervals 1.16–2.83).

Conclusion

Non-smokers with SHS exposure had an attenuated improvement in anginal symptoms compared to those without SHS following EECP.     

Costs and benefits of cervical screening. II. Is it worthwhile reducing the screening interval from 5 to 3 years?

A reduction in screening interval from 5 years to 3 years would greatly increase the cost of the programme, but would save few extra lives. The cost per life saved would be around £250 000 at 1995 prices, or around £8000 per life per year saved. There would in addition be human costs for the women screened. The opportunity cost of reducing the interval may be too great, since it is likely that the Health Service would achieve greater health benefits by investing the funds in other health care activities.     

Investigating UV screening in leaves by two different types of portable UV fluorimeters reveals in vivo screening by anthocyanins and carotenoids

Two portable instruments, designed to evaluate epidermal UV screening in leaves, were compared: the Dualex and the UV-A-PAM fluorimeter. Both instruments excite chlorophyll fluorescence at the same UV wavelengths but reference excitation is in the red and the blue spectral range in the former and the latter fluorimeter, respectively. When analyzing green leaves, general agreement of the data is obtained with the two instruments. In the presence of anthocyanins, the UV-A-PAM fluorimeter provided higher estimates for epidermal UV transmittance than the Dualex fluorimeter, which was attributed to absorption of blue excitation light by anthocyanins. By comparing data from the instruments, anthocyanin-dependent transmittance of 50% was determined in abaxial sides of some autumn leaves, and also in abaxial sides of tropical shade plants. Further, with leaves of chlorophyll b-less mutants of H. vulgare, unusually high epidermal UV transmittance was detected but this was attributed to the lack of chlorophyll b absorption and, in addition, to absorption of blue radiation by xanthophylls which are not functionally connected to photosystems.