Lead optimisation of dehydroemetine for repositioned use in malaria

Drug repositioning offers an effective alternative to de novo drug design to tackle the urgent need for novel anti-malarial treatments. The anti-amoebic compound, emetine dihydrochloride, has been identified as a potent in-vitro inhibitor of the multi-drug resistant strain K1 of Plasmodium falciparum (IC50: 47 nM ± 2.1 nM). Dehydroemetine, a synthetic analogue of emetine dihydrochloride has been reported to have less cardiotoxic effects than emetine. The structures of two diastereomers of dehydroemetine were modelled on the published emetine binding site on cryo-EM structure 3J7A (Pf 80S ribosome in complex with emetine) and it was found that (-)-R,S-dehydroemetine mimicked the bound pose of emetine more closely than (-)-S,S-dehydroisoemetine. (-)-R,S-dehydroemetine (IC50 71.03 ± 6.1 nM) was also found to be highly potent against the multi-drug resistant K1 strain of P. falciparum in comparison with (-)-S,S-dehydroisoemetine (IC50 2.07 ± 0.26 μM), which loses its potency due to the change of configuration at C-1′. In addition to its effect on the asexual erythrocytic stages of P. falciparum, the compounds exhibited gametocidal properties with no cross-resistance against any of the multi-drug resistant strains tested. Drug interaction studies showed (-)-R,S-dehydroemetine to have synergistic antimalarial activity with atovaquone and proguanil. Emetine dihydrochloride, and (-)-R,S-dehydroemetine failed to show any inhibition of the hERG potassium channel and displayed activity on the mitochondrial membrane potential indicating a possible multi-modal mechanism of action. [Abstract copyright: Copyright © 2020 Panwar et al.

Drug repositioning as a route to anti-malarial drug discovery: preliminary investigation of the in vitro anti-malarial efficacy of emetine dihydrochloride hydrate

Background Drug repurposing or repositioning refers to the usage of existing drugs in diseases other than those it was originally used for. For diseases like malaria, where there is an urgent need for active drug candidates, the strategy offers a route to significantly shorten the traditional drug development pipelines. Preliminary high-throughput screens on patent expired drug libraries have recently been carried out for Plasmodium falciparum. This study reports the systematic and objective further interrogation of selected compounds reported in these studies, to enable their repositioning as novel stand-alone anti-malarials or as combinatorial partners. Methods SYBR Green flow cytometry and micro-titre plate assays optimized in the laboratory were used to monitor drug susceptibility of in vitro cultures of P. falciparum K1 parasite strains. Previously described fixed-ratio methods were adopted to investigate drug interactions. Results Emetine dihydrochloride hydrate, an anti-protozoal drug previously used for intestinal and tissue amoebiasis was shown to have potent inhibitory properties (IC50 doses of ~ 47nM) in the multidrug resistant K1 strain of P. falciparum. The sum 50% fractional inhibitory concentration ([n-ary summation]FIC50, 90) of the interaction of emetine dihydrochloride hydrate and dihydroartemisinin against the KI strains of P. falciparum ranged from 0.88-1.48. Conclusion The results warrant further investigation of emetine dihydrochloride hydrate as a potential stand-alone anti-malarial option. The interaction between the drug and the current front line dihydroartemisinin ranged from additive to mildly antagonistic in the fixed drug ratios tested

Selected derivatives of erythromycin B- in silico and anti-malarial studies

Erythromycin A is an established anti-bacterial agent against Gram-positive bacteria, but it is unstable to acid. This led to an evaluation of erythromycin B and its derivatives because these have improved acid stability. These compounds were investigated for their anti-malarial activities, by their in silico molecular docking into segments of the exit tunnel of the apicoplast ribosome from Plasmodium falciparum. This is believed to be the target of the erythromycin A derivative, azithromycin, which has mild anti-malarial activity. The erythromycin B derivatives were evaluated on the multi-drug (chloroquine, pyrimethamine, and sulfadoxine)-resistant strain K1 of P. falciparum for asexual growth inhibition on asynchronous culture. The erythromycin B derivatives were identified as active in vitro inhibitors of asexual growth of P. falciparum with low micro-molar IC50 values after a 72 h cycle. 5-Desosaminyl erythronolide B ethyl succinate showed low IC50 of 68.6 µM, d-erythromycin B 86.8 µM, and erythromycin B 9-oxime 146.0 µM on the multi-drug-resistant K1 of P. falciparum. Based on the molecular docking, it seems that a small number of favourable interactions or the presence of unfavourable interactions of investigated derivatives of erythromycin B with in silico constructed segment from the exit tunnel from the apicoplast of P. falciparum is the reason for their weak in vitro anti-malarial activities

Successful Protein Extraction from Over-Fixed and Long-Term Stored Formalin-Fixed Tissues

One of the major breakthroughs in molecular pathology during the last decade was the successful extraction of full-length proteins from formalin-fixed and paraffin-embedded (FFPE) clinical tissues. However, only limited data are available for the protein extraction efficiency of over-fixed tissues and FFPE blocks that had been stored for more than 15 years in pathology archives. In this study we evaluated the protein extraction efficiency of FFPE tissues which had been formalin-fixed for up to 144 hours and tissue blocks that were stored for 20 years, comparing an established and a new commercial buffer system. Although there is a decrease in protein yield with increasing fixation time, the new buffer system allows a protein recovery of 66% from 144 hours fixed tissues compared to tissues that were fixed for 6 hours. Using the established extraction procedure, less than 50% protein recovery was seen. Similarly, the protein extraction efficiency decreases with longer storage times of the paraffin blocks. Comparing the two buffer systems, we found that 50% more proteins can be extracted from FFPE blocks that were stored for 20 years when the new buffer system is used. Taken together, our data show that the new buffer system is superior compared to the established one. Because tissue fixation times vary in the routine clinical setting and pathology archives contain billions of FFPE tissues blocks, our data are highly relevant for research, diagnosis, and treatment of disease

Homeostasis in dynamic self-regulatory physiological systems

Understanding the general principles of homeostasis and its regulation in health and disease is key to managing patients in intensive care units and operating theatres. In these environments, it is crucial to realize that physiological control is a dynamic process aimed at achieving a balance between two opposing sets of factors. Whereas one set of factors (e.g. the sympathetic nervous system) attempt to increase a physiological variable of interest at any given time, opposing forces acting almost concurrently (e.g. the parasympathetic nervous system) will result in the reduction in the value of this variable. The human body is a self-adapting system and as a result of its ability to adapt, new physiological ‘steady states’ will be reached and maintained even in diseases. This review will explore some of the concepts and pathways involved in the regulation of homeostasis in the immediate, intermediate and delayed time scales following an initial perturbation, emphasizing the dynamic nature of this regulation

Hormonal control of metabolism : regulation of plasma glucose

Blood glucose concentrations are required to be maintained within a narrow therapeutic range in order to ensure the normal functioning of the body. This is accomplished through a complex, interactive, finely coordinated neuro-endocrine regulatory process. Hormonal control through the opposing actions of insulin and glucagon secreted by the islet cells of the pancreas serve as the primary response mechanism to avert post-prandial hyperglycaemia and fasting hypoglycaemia. In addition to this basic response, a range of endocrine mediators concurrently intervene, to enable the fine modulation of the process through a range of insulin-dependent and insulin-independent processes, which ultimately achieve glycaemic control by influencing tissue glucose uptake, glycolysis, glycogenesis, glycogenolysis and gluconeogenesis. More recent evidence supports a central, predominantly hypothalamic role initiated through nutrient (glucose, fatty acid) and hormonal (insulin, leptin, glucagon-like peptide-1) stimuli that influences glucose regulation by direct or indirect effects on skeletal muscle glucose uptake, islet cell insulin/glucagon secretion and hepatic glucose production

Co-creation and empowerment: pathways to better student engagement

Progression and employability are at the forefront of current conversations in higher education, with much effort invested in developing complex interventions to improve graduate outcomes. Pedagogy research indicates that factors such as a sense of belonging, impostor syndrome and a meaningful connection to campus life are contributing factors to student success. Furthermore, student outcomes correlate closely with socio-economic status and demographic data. Across all disciplines, academic teams work tirelessly to “close the gap” created by social inequalities, but it remains to be determined where efforts are best placed. For instance, many universities offer access programmes to low-participating neighbourhoods, while other institutions place great effort on the BTEC and BAME attainment gaps. Here we share guidance on effective student engagement based on our anecdotal experience and propose co-creation with and empowerment of all students as a desirable alternative pathway to success

Mining the archival formalin-fixed paraffin-embedded tissue proteome: opportunities and challenges

The significant potential of tissue-based proteomic biomarker studies can be restricted by difficulties in accessing samples in optimal fresh-frozen form. While archival formalinfixed tissue collections with attached clinical and outcome data represent a valuable alternate resource, the use of formalin as a fixative which induces protein cross-linking, has generally been assumed to render them unsuitable for proteomic studies. However, this view has been challenged recently with the publication of several papers accomplishing variable degrees of heat-induced reversal of cross-links. Although still in its infancy and requiring the quantitative optimisation of several critical parameters, formalin-fixed tissue proteomics holds promise as a powerful tool for biomarker-driven translational research. Here, we critically review the current status of research in the field, highlighting challenges which need to be addressed for robust quantitative application of protocols to ensure confident high impact inferences can be made