Mapping the Streptomyces Genome
A Critical Step in Learning How to Make Nikkomycin Z
As a Possible Cure of Valley Fever

Valley Fever (coccidioidomycosis) is a serious problem for both humans and pets. Fortunately, many Valley Fever infections go away without serious consequences. However, for others infection progresses to cause chronic pneumonia, osteomyelitis, skin abscesses, and meningitis. These complications are often life-threatening without treatment. The current treatments available are valuable in controlling symptoms and even healing the sites of infection. However, they do not always work and none actually cures the infection. For humans, this means that complications often take many years to control and for pets, the illness can be so debilitating that they must be euthanized. Clearly there is a need for advances in treatment against Valley Fever.

Nikkomycin Z is a drug made by a particular bacteria, named “Streptomyces tendae”. This bacteria, which for simplicity we will refer to simply as “Strep,” was first discovered to have antifungal properties in the 1970’s by scientists at the German pharmaceutical company, Bayer IG. In the 1980’s a California microbiologist, Richard Hector, discovered that nikkomycin Z was effective in treating experimental Valley Fever infections in mice. Very importantly, Dr. Hector observed that nikkomycin Z treatment eradicated the infection in mice rather than just suppressing it as was the case with other drugs. In the 1990’s a company in the San Francisco bay area, Shaman Pharmaceuticals, sponsored a program to study the use of nikkomycin Z in the treatment of Valley Fever. However, by 2000 this company went out of business and liquidated its assets. A foundation associated with California State University at Bakersfield acquired the rights to nikkomycin Z and thus saved it from disappearing. However, it was not until May of 2005 when this foundation transferred the rights to the Valley Fever Center for Excellence at the University of Arizona that further development was restarted.

The immediate goals of the Nikkomycin Z Development Project are to 1) restart clinical trials in humans and 2) develop an inexpensive method of producing more nikkomycin Z. Clinical trials have now restarted with the help of research grants from the NIH ($625 thousand), and the FDA Office of Orphan Product Development ($1 million) and a charitable gift from the JT Tai and Company Foundation ($300 thousand). Restarting clinical trials was also possible because there already existed nearly 3 killograms of nikkomycin Z made by Shamam Pharmaceuticals which was suitable for human use. As a result, a small safety study was initiated in the fall of 2007. However, when this study is completed in the next three years, there will remain no further drug available for future studies in either humans or pets and it is for this reason that a new method for making more drug is critical if the project is going to continue.

Making nikkomycin Z.

How drugs are made. Making drugs like nikkomycin Z are typically done one of two ways. One approach is to simply make the drug through a series of chemical reactions and purifying the synthesized drug from other impurities. This is called synthetic manufacturing. A second approach where the drug is originally discovered to be made by a microorganism (as in the case with nikkomycin Z) is to improve on this discovery by finding ways to induce the bacteria to continue making the drug and then purify it from other impurities. This is called fermentation manufacturing because fermentation is the process of bacterial growth. Occasionally the two approaches can be mixed in precesses called semi-synthetic manufacturing. For the nikkomycin Z project, we are pursuing both synthetic and fermentation approaches to develop our manufacturing process.
The synthetic approach has never been done before and therefore is less certain to work. However, if successful, it could offer several advantages including low cost and simplicity.
The fermentation approach is how the early manufacturing of nikkomycin Z was carried out. Because this approach has already been successful, we know it can be done again. However, in the past the manufacturing process was very expensive. For example, a single kilogram of nikkomycin Z in the 1990’s cost about $70 thousand dollars to manufacture and so the cost of drug alone for the small clinical study now underway was about $200 thousand or over $3,000 per subject. It would almost certainly cost many time this much to manufacture the same way today, a cost that is prohibitively expensive to move the project forward. It is for this reason that we need to develop a new less expensive manufacturing process.

Improving the fermentation process of nikkomycin Z production. Our plan for improving the fermentation process consists of altering the genetic make-up of the drug-producing Strep bacteria to result in two changes: reduce the amount of contaminants that the bacteria makes and increase the amount of drug makes while it grows.
Reducing contaminants. One of the major contaminants that Strep makes is a related drug, nikkomycin X. Both nikkomycin Z and nikkomycin X are made through a series of chemical changes that are controlled by different sets of genes in the bacterial genome. We know which genes needed for making each drug. Some genes are needed to make both drugs and other genes are needed for only one or the other drug. Knowing this, it would make sense that eliminating a gene from the bacterial genome that was needed to make nikkomycin X, we would eliminate the production of nikkomycin X and thereby eliminate a major impurity from the process. We have been working on this approach over the past year with some encouraging success.
Getting the bacteria to produce more nikkomycin Z. This approach was studied thirty years ago by Bayer IG and several stains were developed that indeed made greater amounts of nikkomycin Z as they grew. The scientists at Bayer accomplished this by exposing the bacteria to conditions such as ultraviolet light or chemicals that damaged or changed the DNA in the bacterial genenome. These changes occurred all over the genome resulting in a wide variety of effects in different bacteria. By studying different bacteria, the Bayer scientists identified certain ones that were better producers than the starting strain. However, along with this improvement, it was also discovered that over time these strains stopped producing nikkomycin Z altogether and so their use as a producing strain for a manufacturing process was somewhat limited.

How the Strep Genome Project will help us make nikkomycin Z.

The tools available to understand how the bacterial genome influences drug production have undergone immense improvements since the Bayer scientists conducted their work. For example, they only could induce random changes in the DNA, a very blunt approach to altering the genome. In contrast, it is now possible to make precise changes at exactly the place in the genome that a change is desired. This precision allows the desired changes to be made without potentially deleterious changes to creep in. However, in order to use such precise tools, it is necessary to know exactly where to make the changes. Fortunately, the strains previously made by the Bayer scientists are available to us today. By learning what changes were made by chance, we should know range of genome changes that be responsible for the favorable drug producing characteristics. Also, patterns of genome changes that are similar between the This can be done by comparing the total genome of the producing strains with the parent and noting the difference. This is where the Strep genome project comes into play.
DNA sequencing became front-page news with the human genome project a decade ago. At the time it was first conceived, the human genome project was enormously ambitious, partly because the human genome is very large and partly because the cost of DNA sequencing was relatively expensive. Since then, technologic advances have resulted in dramatic reductions in the cost of sequencing so that there is now discussions about everyone sequencing their own genome in the foreseeable future. We are not quite there yet: current cost would be over $1 million per person! However, with respect to genomes of bacteria such as Strep, they are much smaller, less than 1% than the human genome. Thus the challenge of sequencing strains of Strep is now well within reach. At the Bio5 Institute of the University of Arizona, there now exists the capacity to carry-out the needed sequencing rapidly (within weeks) and feasibly.
The objective of the Strep Genome Project is to sequence the entire genome of the original stain of Strep that was found to produce nikkomycin Z. Additionally, the Project will sequence three high-producing strains derived from the original strain. Comparison of the three strains to the parent and to each other will allow the identification of changes from the parent that are common to the high-producers. Similarities in changes among the high-producers as compared to the original strain would provide strong evidence of genome changes responsible for the high-production characteristic common to those strains. This information will be used to direct targeted genome modification in the original strain. By this means we hope to create a new high-producing strain but one without the damage that might have limited the usefulness of the earlier Bayer strains.

Funding the Strep Genome Project.

Since the fall of 2006, many dog-lovers across the state of Arizona and elsewhere in the country have generously supported the Nikkomycin Z Development Project because of its potential value in saving the lives of their pets and those of others. These funds were not explicitly directed at supporting any one aspect of the drug development project activity and have continued to accumulate. As of September, 2007, donations from nearly 150 pet owners have totaled approximately $33,500. Much of the effort for coordinating this fund-raising was contributed by Shirley Cole and Janice Arenofsky of Scottsdale, Arizona. Also, single generous contribution of $10,000 was provided by Patricial and Bruce Bartlett of Rancho Sante Fe, California. This generous support from such a large number of like-thinking persons deserves to be identified for its specific contribution to the overall program to develop nikkomycin Z. It would seem fitting that the Step Genome project would be just such a component to designate as contributed by pet-owners.
The total cost of the Strep Genome Project is estimated to be $70 thousand. The original strain will cost approximately $28 thousand and each of the three derivative strains will cost $14 thousand each to sequence. With contibutions already made, we are in a position to complete the sequencing of the parent strain now and as future funds become available we will proceed to sequence the three derivative strains.
We hope that full funding of the Strep Genome Project can be found from the broad pet-lover community which might also include companies that market to pet-owners as well as from the owners themselves. If so, it would certainly be appropriate to modify the “Strep Genome Project” in order to give recognition to the source of support that made the project possible. Discussions about this possibility are currently underway.