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UCSD Team Taps MSH Synthesis Pathway To Develop Drugs for MDR Tuberculosis

Robert Fahey, a research professor of chemistry and biochemistry at the University of California, San Diego, won a three-year, $320,000 R01 grant from the National Institutes of Health that began on April 1. Fahey and his team are looking for compounds that block the synthesis of mycothiol, also known as MSH, the major thiol present in actinomycetes.
MSH has functions similar to those of glutathione. MSH has been shown to be essential for the growth of Mycobacterium tuberculosis, and is thought to be necessary for its persistence in the dormant state.
These compounds will serve as leads for the development of new drugs for multi-drug resistant tuberculosis and as tools to help investigate the biological functions of MSH in the many species of actinomycetes in which it is found.
High-throughput screening assays will be developed to identify inhibitors of the MshA and MshC enzymes and inhibitors that block MSH synthesis in intact M. smegmatis cells.
What is the duration and purpose of this grant?
It is a three-year grant to develop screening methods to identify inhibitors of MSH biosynthesis as potential anti-TB drugs, and then apply them at the NIH screening facilities where they have large compound libraries.
Do you have a timeline for the different phases of this project?
We are just about to order the equipment that we’ll need to do this on a large enough scale to allow us to move it onto the NIH screening facilities.
We have done the preliminary studies that formed the basis for the grant proposal. Now we have to do the detailed studies to prepare for doing HTS on a large scale. We hope to be ready within a year to move the screening assays into HTS.      
Could you give me some background on your work?
About a dozen years ago, we discovered that a novel thiol, mycothiol, is produced by all actinomycetes, including the pathogens Mycobacterium tuberculosis, M. leprae, Corynebacterium diphtheriae, bacteria of the genus Streptomyces, and other antibiotic-producing actinomycetes as well.
Our group and two other groups pretty much elucidated the structure of MSH. MSH is a low-molecular-weight thiol (mass 486), which comprises an N-acetylcysteine amide linked to a glucosamine residue that is itself alpha (1-1) linked to myo-inositol.
MSH has functions similar to those of glutathione. A lot of attention has been focused on glutathione in recent years as a protectant against oxidative stress and toxins of all kinds.
We found that gram-positive bacteria do not synthesize glutathione. At that time, it was thought that glutathione was essential for life. So this discovery came as a big shock to us. The question became, “How do these gram-positive bacteria survive if they do not make glutathione?”
Actinomycetes apparently survived by having evolved the biosynthesis of MSH. This goes back in evolutionary history to before oxygen accumulated in the environment. Gram-positive and gram-negative bacteria both evolved different mechanisms for dealing with oxygen toxicity based on a thiol derived from cysteine.
We have focused on M. tuberculosis, because of the importance of developing new approaches to anti-TB drugs. Our approach has been to look at the biosynthetic pathway, the idea being that knocking out the synthesis of MSH may sensitize and make it unable to survive in humans.
We have done knockouts of genes previously identified in M. tuberculosis as being a part of the MSH biosynthesis pathway. We demonstrated that two of the genes involved in the pathway, designated mshA and mshC, are essential for the growth of M. tuberculosis.
We identified these genes in a model organism, M. smegmatis, which is easier to work with compared to M. tuberculosis because it is non-pathogenic and fast-growing. We were able to show that if we knock out the mshA and/or the mshC genes, MSH biosynthesis is completely blocked.
We have also done knockouts of two other genes, mshB and mshD, and found that inactivating these genes does not completely prevent MSH biosynthesis and M. tuberculosis growth.
Our focus is on the MshA and MshC enzymes as possible targets for anti-TB drugs. We have been developing high-throughput screens for detecting these targets, and an assay to determine if an inhibitor will block MSH biosynthesis in intact cells.
We grow the cells in the presence or absence of a potential inhibitor, and measure their optical density to determine if they are growing in the presence of the compound. The bacteria we use for this are M. smegmatis, because we know that they will grow even though MSH biosynthesis is blocked. [The mshA and mshC genes of MSH biosynthesis that are essential for the growth of M. tuberculosis are not required for the growth of M. smegmatis.]
After the bacteria have grown up to a density that corresponds to an A600 value of about 1, we add monochlorobimane, a fluorescent labeling reagent for thiols. This reagent penetrates cells and reacts with MSH to form an S-conjugate. The S-conjugate is a substrate for an enzyme that is very active in the cells. This enzyme cleaves the S-conjugate in half.
The acetylcysteine portion, which is also an S-conjugate, is released from the cells. This S-conjugate is a mercapturic acid. Mercapturic acids are a way that M. smegmatis excretes toxin. This mercapturic acid is also highly fluorescent, unlike monochlorobimane.    
This allows us to treat cells with monochlorobimane, and after a one-hour incubation, [we] read the fluorescence in the solutions containing the cells in a Tecan M200.
The degree of fluorescence is related to the amount of MSH in the cells. This provides an assay for the identification of MSH biosynthesis inhibitors in whole cells.  
In this one assay, we can evaluate the ability of the inhibitor to get into the cell and its inhibition of any enzymes in the pathway that will block MSH production. The question becomes, “If you get a positive hit, what target is it hitting?”
We have specific enzyme assays and assays to detect metabolic intermediates and such in the cells. We can use these assays to identify which target is hit by a given inhibitor.
Is this technology something that you may commercialize?
We have patents, either awarded or pending, on all of the targets that we have identified and our methodology.
Who would be the primary customers for this technology?
Pharmaceutical and drug-discovery companies. The difficulty in finding a licensee for the patents is that TB is not a very prevalent disease in the US or other developed countries. It occurs mainly in underdeveloped countries where the market for expensive drugs is not very good. But the NIH is putting a lot of money into the development of anti-TB drugs.
A global alliance is now linking a number of different resources to fund the development of anti-TB drugs, but it will all be based on, presumably, minimizing the cost of supplying the drugs.
Some pharmaceutical companies have expressed an interest in developing anti-TB drugs, but they are not doing it to make a profit in the places where the drugs will actually be used. I am not quite sure why they are doing it, except that maybe they think the lost profits can be recouped in the smaller but more lucrative markets for TB drugs in developed countries. Or they feel that the highly drug–resistant (XDR) strains of TB may get released into populated countries. Then expensive anti-TB drugs may be marketable.
Why is there a big push to fund anti-TB drug development?
It started back in the early 90s when there was a rash of drug-resistant TB outbreaks in a number of places in the US, including New York City.
The outbreak in New York prompted the NIH to start putting money into anti-TB drug development in the early 90s, and the current and still important impetus is the threat of XDR strains of TB.
In Africa, whole villages are being essentially wiped out by XDR TB. If XDR strains of TB ever got out of Africa and into developed countries, it would be a significant problem. And you have to be prepared to deal with it in advance. You cannot depend on developing the drugs for it after it has already started a major epidemic.
Is TB a problem among HIV-positive persons and those with AIDS in developed countries?
Approximately 40 to 50 percent of HIV-positive deaths in Africa are caused by TB. It is less of a problem among those with HIV infection and AIDS in the US, but is somewhat of an issue along our borders. For example, San Diego sees a much higher percentage of TB patients than cities in the northern parts of the US.
But TB is a relatively rare problem in the US. It is just the scare of XDR strains of TB and the problem in underdeveloped countries that is pushing drug-development efforts.

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