Waging Warfarin against Tuberculosis: A Review of Dutton et al, 2010

A review of Dutton et al. Inhibition of bacterial disulfide bond formation by the anticoagulant warfarin. PNAS (2010) vol. 107 (1) pp. 297-301 PMCID: PMC2806739

This PNAS study from a few years ago looks at the potential antibacterial use of a popular anticoagulant, warfarin. Although my attention was drawn to this article at first through a journal club, I think that this is an interesting paper to review for several reasons. First, it is a paper published in a relatively high profile journal, and it also note worthy because it is research that has a more direct connection to clinical applications and drug development. 

This work has also been reviewed by other blogs and science writers (in a more timely fashion than I have done). For example, see a guest post in Small Things Considered blog written by two graduate students entitled "All is fair in love and warfarin". These students do a good job of summarizing the article, especially for a wider audience. A more in-depth review (and slightly more critical) entitled "Warfarin: Not Just Rat Poison" is also worth a read.

Here, I would like to add my commentary to this work, going further than the above two reviews / summaries in pointing out some flaws. The very rationale of this work is questionable, and might provide a glimpse into the misalignment of public interest and the motivation of the scientists. This review also happens to continue an unwitting trend of reviews on anti-tuberculosis drug papers, following my review of Shi et al, 2011, entitled "Grainy Westerns and Fuzzy Logic".

Manuscript Highlights

1. High doses of warfarin (and lower doses of the anticoagulant phenindione) are capable of inhibiting the growth of Mycobacterium smegmatis and tuberculosis (only warfain data for M. tb)

2. Mycobacterium VKOR, the homolog of the human target of Warfarin, is active when expressed in E. coli. The activity of this enzyme in these experiments is sensitive to Warfarin.

3. Surprisingly, inhibition of Mycobacterium VKOR by warfarin does not appear to be the reason why this drug inhibits M. tuberculosis growth. Should either VKOR or Warfarin really be the focus of drug development?

Synopsis:

In this study, Dutton and colleagues explore the use of the anticoagulant warfarin to inhibit the Mycobacterium tuberculosis VKOR enzyme (MtbVKOR). This work could lead to the development of novel, VKOR-targeting anti-tuberculosis therapeutics. This bacterial enzyme, MtbVKOR, is the ortholog of the human target of warfarin, Vitamin K epOxide Reductase (VKOR). While human VKOR is involved in the clotting pathway and bacterial VKOR is thought to be responsible for intracellular disulfide bond formation, the underlying enzymatic activity for these orthologs are very similar. In a previous study, the authors demonstrated that MtbVKOR is active in disulfide bond formation when expressed in E. coli, thereby providing a convenient system for studying the properties of this enzyme in vivo.

Utilizing this heterologous E. coli / MtbVKOR expression system, the authors demonstrate that high doses of warfarin can inhibit the activity of the Mycobacterium enzyme in this setting. This demonstrates that the bacterial and human VKOR enzymes share more than just some sequence similarity. Selection and analysis of warfarin-resistant variants of MtbVKOR supports this conclusion. Finally, the authors demonstrate that warfarin has antibacterial properties against Mycobacterium species, although they cannot identify VKOR as the target in this case.

Select 'Read More' to see the rest of the review.

Building a Better Future, One Base at a Time

Synthetic Biology holds promise to revolutionize biotechnology and many other industries. Broadly defined, it is an engineering approach that seeks to design artificial genes, gene regulatory circuits, genomes, and useful and novel cellular behaviors. This includes efforts in recent years to program bacteria to specifically invade tumor cells as well as the production of microbial derived artemisinin. In addition to these impressive feats of bio-engineering, synthetic biology also gives researchers a way to better understand the design principles by which normal cells function. After all, it is much easier to understand how a system works once you have succeed in constructing one of similar complexity.

The scale of gene construction involved in synthetic biology can be quite large. In a multi-million dollar effort by the J. Craig Venter Institute, researchers successfully synthesized an entire bacterial genome and transplanted it into a cell. While the synthetic genome contained mostly natural sequence (the genome was physically synthetic, but the information contained within was a copy of what nature and evolution has already produced), future efforts may involved rewriting of vast sections of the genome. These grand examples of synthetic biology highlight a major obstacle facing this emerging discipline: the costs involved in the actual de novo synthesis and assembly of DNA.

Over the past decade and a half, tremendous advances in DNA sequencing technology have been made. The original human genome project took years of efforts by many researchers, with a final price tag estimated at approximately 4.4 billion dollars (current dollars, adjusted for inflation) or slightly more than $1 per base pair. Due to the improved scale of sequencing coupled with several technological revolutions, scientists are rapidly approaching the coveted $1000 genome, which would be approximately a million fold improvement in terms of cost (or $0.000001 per base)

DNA synthesis technology and capabilities currently sit where DNA sequencing was a decade ago, when the completion of the final draft of the human genome was announced. As a matter of fact, synthesis technology is arguably behind even this mark. The Venter Institute's creation of Mycoplasma laboratorium (also known as Mycoplasma genitalia JCVI-1.0) is estimated to have taken a small team of researchers nearly a decade and $40 million dollars to complete. Since this genome is only half a million dollars, this represents a cost of $80 a base pair. Most smaller projects demand less intense and less iterative assembly efforts, but synthesis of even short oligos is still between $0.25 and $1.0 per base, depending on the size, scope, and quality of the synthesis efforts.

Clearly, there is room for improvement in DNA synthesis technology. This improvement will be critical to the advance of the field of synthetic biology, which hold tremendous promise for a variety of industries. Where will the improvement come from? For DNA sequencing, improved scale and efficiency of existing technology was important, but not enough for the incredible leap in capability and cost-effectiveness. Both evolution and revolution (innovative second and third generation technologies) was necessary to achieve the current level of capability. 

So far, the horizon for new, ground-breaking technology for DNA synthesis is not clear. The chemistry used in the synthesis of oligomers has remained largely unchanged for years. There are a few new approaches which may hint at how DNA synthesis can be made more reliable and affordable. Here, I'll survey a few of the newest developments. This includes the MOSIC method, the research by Dr. George Church's group at Harvard and its implementation at Gen9, as well as Cambrian Genomics and some other creative ideas. 

Select 'Read More' to learn more about emerging DNA synthesis technologies.