Thursday, August 28, 2014

Focus on: Novel tuberculosis treatments

Tuberculosis (TB) is a widely prevalent disease, causing millions of deaths across the world. Although it is treatable, this is difficult and requires administration of multiple antibiotics. There has recently been a growing problem associated with the appearance of resistant strains. There is also a growing problem of tuberculosis amongst cattle in the UK, with the recent badger cull an attempt to address the transmission of TB to cattle.

Scanning electron micrograph of Mycobacterium
tuberculosis 
bacteria, which cause TB


One of the traditional classes of antibiotics known as caprazamycins target the enzyme MraY, which is involved in the production of peptidoglycans, an integral part of the bacterial cell wall. Some caprazamycin resistant strains are known to overexpress MraY, making MraY expression and activity an important area of research. A recent research paper published in the Journal of Biological Chemistry 1 describes an approach where a caprazamycin derivative was created that is effective against strains overexpressing MraY as it targets WecA, an enzyme involved in the synthesis of an integral part of the cell wall structure. WecA could therefore be a promising novel target for a new class of antibiotics in the fight against antibiotic resistant strains of Tuberculosis.



In addition, BMG LABTECH recently released an application note for a FRET screening approach to identify modulators of MraY activity entitled: A high-throughput, homogeneous, FRET-based assay to detect bacterial membrane-bound enzyme (MraY) activity

  1. Y. Ishizaki, et al 'Inhibition of the First Step in Synthesis of the Mycobacterial Cell Wall Core, Catalyzed by the GlcN Ac-1-phosphate Transferase WecA, by the Novel Caprazymycin Derivative CPZEN-45' J. Biol. Chem 2013, 288:30309-30319


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Tuesday, August 26, 2014

Application: High throughput screening assay to identify anti-malarial drugs

Malaria is a parasitic disease spread through mosquitos. It is widespread in many areas with poor access to good quality medical care, and is one of the most devastating diseases on the planet, often leading to coma or death. In fact, the World Health Organisation estimate that in 2010, the disease killed between 660,000 and 1.2 million people (1). Although anti-malarial drugs are available, resistance to these is increasing, with potential impact on the health of millions of people, as well as having a major negative effect on the economies of those countries affected.

A family of ATP dependent plasma membrane pumps have been implicated by Genome Wide Association Studies to have roles in a variety of different conditions. One particular member of this family (PMCA4) has been shown to be associated with resistance to malarial infection. Due to the wide ranging effects of the PMCA family, a group in Manchester devised an assay to screen for potential drugs targeting this group of proteins, using PMCA4 as a model (2).

To do this, they used the CisbioTranscreener assay which uses FRET technology in combination with long lived fluorophores to assay for ATPase activity with a Z’ of greater than 0.5.  Using this assay, over 20,000 compounds were screened in a single day using the FLUOstar OMEGA, leading to nearly 1500 compounds that inhibited PMCA4 activity by more than 50%.
The Cisbio Transcreener assay used to identify drugs that target PMCA4
was detected with the FLUOstar OMEGA from BMG LABTECH

Although it is very early days in the drug discovery process, someday a descendant of one of these compounds may have an application as an anti-malarial treatment, bringing potential relief to millions of people.

  1.  NayyarGML, Breman JG, Newton PN, Herrington J (2012). "Poor-quality antimalarialdrugs in southeast Asia and sub-Saharan Africa". Lancet InfectiousDiseases 12 (6): 488–96.
  2. Mohamed TM, Zakeri SA, Baudoin F, Wolf M, Oceandy D, Cartwright EJ, Gul S, Neyses L (2013). “Optimisation and validation of a high throughput screening compatibleassay to identify inhibitors of the plasma membrane calcium ATPase pump--anovel therapeutic target for contraception and malaria”. J Pharm Pharm Sci 16 (2): 217-30

Thursday, August 21, 2014

Applications: Monitoring Calcium Flux in 3D Heart Tissues in Real Time

In order for a drug to attain approval for use it must not only be shown that it has the desired effect but that it does not have undesirable side-effects. Traditionally, laboratory animals are used to predict possible side-effects but the animals may respond differently than humans so an effective in vitro system is highly desirable.

In order for an in vitro system to be most useful it should best mimic the in vivo tissue. For this reason 3D cell culture approaches have become more prevalent and have proven to exhibit fewer misleading results than their 2D counterparts.

The scientists at InvivoSciences have recognized the importance of using 3D cultures for drug assessment and have met the challenge of creating a suitable in vitro system with their Mini-Construct ChambersTM .

The CLARIOstar® captures periodic Ca2+-transients 
that induce cardiac contractions

In an application note now available on the BMG LABTECH website InvivoSciences scientists describe the use of the CLARIOstar® to monitor the function of engineered heart tissues. A calcium indicator was used to monitor the calcium transients that induce cardiac contractions. The ability of the CLARIOstar® to read 100 data points per second allow it to measure the rapid changes in fluorescence intensity that are associated with the calcium flux from a beating heart.

Together, BMG and InvivoSciences can provide a platform for the analysis of drug effects on heart function and do so at the early of stages drug discovery!

BMG LABTECH Application Note 253: Real time calcium flux measurements in iPSC derived 3D heart tissues.

Tuesday, August 19, 2014

New software enables detection of disease associated insertion deletion mutations

Insertion deletion mutations (indel mutations or indels) can have a profound effect on biological function. As their name suggests this is when genetic sequence is either added or removed.  The effect of these mutations is especially evident when these mutations occur in a protein coding region as they can lead to the production of proteins that are truncated or otherwise altered so that function is severely impacted. However, these indel mutations can vary in length, from 1 base pair to thousands. Furthermore the sequence that is inserted or deleted can be highly variable.The variability of indels has made them difficult to detect within the genome, until now!

Photograph of CSHL in the fall of 2008
by AdmOxalate
A group of scientists at Cold Spring Harbor Labs have developed and used a new software program called Scalpel to precisely see where indels are causing cuts in the genome. The results of their work are published in a recent issue of the journal Nature Methods in an article entitled: 'Accurate de novo and transmitted indel detection in exome-capture data using microassembly'.



The group used Scalpel on a genome database produced from families in which one child suffered from autism and the rest of the family is unaffected. This approach allowed them to identify indels that likely disupt genes in autistic children.

The scientist are now seeking to apply Scalpel more broadly and are collaborating with any scientists, such as cancer biologists, that are looking for indels.

Some material for this post was obtained from the Science Daily article: A shift in the code: New method reveals hidden genetic landscape

Original Article citation: Giuseppe Narzisi, Jason A O'Rawe, Ivan Iossifov, Han Fang, Yoon-ha Lee, Zihua Wang, Yiyang Wu, Gholson J Lyon, Michael Wigler, Michael C Schatz. Accurate de novo and transmitted indel detection in exome-capture data using microassemblyNature Methods, 2014

Thursday, August 14, 2014

Applications: A novel ultrahigh-throughput fluorescence anisotropy-based assay for ATP-competitive inhibitors of TilS

TilS (tRNAIle lysidine synthetase) is an essential, ATP-dependent enzyme which is conserved in bacterial pathogens. The high degree of conservation of TilS among pathogenic bacteria combined with its absence in human nuclear and mitochondrial genomes make it an attractive potential target for novel antibacterial drugs.

E. coli
With this in mind scientists at AstraZeneca created a screening platform which is described in the recent Journal of Biomolecular Screening article entitled: ‘Discovery of ATP-Competitive Inhibitors of tRNAIle Lysidine Synthetase by High-Throughput Screening’. To enable this screening these scientists used ATP linked with either the fluorophore BODIPY or TAMARA. Binding of the labeled ATP to TilS enzymes from E. coli and S. aureus could be measured by an increase in fluorescence anisotropy and treatment with an ATP-competitive inhibitor would displace the labeled ATP leading to a decrease in fluorescence anisotropy.

We at BMG are happy that our PHERAstar HTS microplate reader could be used in this novel screening approach!


For more information on the PHERAstar and other BMG LABTECH microplate readers please visit our website: www.bmglabtech.com