What could be the explanation for a drug that works at lowdoses to help children with one type of early puberty butrequires doses ten times higher to treat a similar disease?For years, this puzzle has intrigued pediatricendocrinologists, the doctors who care for children withabnormal hormone systems.
Now scientists at UC San Francisco, working with a newexperimental model that turns yeast cells into littleversions of human adrenal glands, have learned the answer:they have shown how the drug, medroxyprogesterone acetate(MPA) inhibits one of the key enzymes necessary to producesteroid hormones.
MPA more commonly is known by its brand name, Provera. Oftenprescribed as a hormone replacement therapy duringmenopause, it sometimes is used to treat small children whodevelop breasts, enlarged testicles and other sexcharacteristics. In high doses it is used to treat womenwith breast cancer.
The research, published in the June issue of The Journal ofClinical Endocrinology and Metabolism, was conducted bysecond year Tufts University medical student Tim Lee andUCSF research scientist Richard Auchus, MD, PhD, in thelaboratory of Walter L. Miller, MD, UCSF professor ofpediatric endocrinology. The study, conducted on a summerresearch grant from the Society for Pediatric Research(SPR), earned Lee a prestigious Medical Student ResearchAward at the annual meeting of the Society for PediatricResearch in May.
Lee and Auchus were looking for a way to explain MPA's dualmechanism of action. Normally, puberty starts in thepre-teen years, when certain cells in the brain beginpulsing out small timed doses of a chemical signal calledGnRH, for gonadotropin releasing hormone. GnRH instructs theovaries and testes to start making steroids, including sexhormones. For children who start to develop sexcharacteristics very early as young as toddler age Provera is one of a number of drugs that can be used todelay the onset of puberty. Scientists already have shownthat small amounts of the drug bind to receptors in thebrain and inhibit GnRH production.
However some children have a condition calledgonadotropin-independent sexual precocity. Though theirbrains are not yet pulsing out GnRH, their ovaries andtestes begin making steroid hormones anyway. They havedisorders such as McCuneAlbright Syndrome, which causesovarian cysts in young girls, and testotoxicosis, whichcauses early masculinization in young boys. Because thesechildren's bodies are making steroid hormones without a GnRHsignal from the brain, low doses of MPA are ineffective.Instead, UCSF pediatric endocrinologists have successfullytreated these children with higher doses of the drug. Thesuccess of the treatment proved that larger doses of MPAmust interfere with some step of hormone production otherthan GnRH.
Miller and his lab study the process that the body uses toproduce steroid hormones. Sex steroids are produced when theovaries and testes convert cholesterol into androgens,estrogens and related substances. The process takes severalsteps, and each step is mediated by a different enzyme, aprotein that speeds and guides a biological process. WithAuchus' guidance, Lee was assigned to investigate whetherMPA works at high doses by blocking the action of one ofthese enzymes.
Studies of rats had shown that large doses of MPA mightblock the action of cytochrome P450c17, a key enzyme at workin the conversion of cholesterol into hormones. But when Leeand Auchus tested MPA on the human form of P450c17, theenzyme's action was unaffected. "This emphasizes a point:rats are not always a good model for human biology," Millersaid.
Lee and Auchus next tested MPA on 3bHSDII (3-beta-HSD-2),the next step after P450c17 in the cascade of enzymesessential to making human hormones. They found that MPAinhibits 3bHSDII by binding to the site that the enzyme usesto help synthesize hormones. They also showed that thisaction is dose-related their biochemical data corroboratedphysicians' clinical experience that relatively high dosesare needed for a therapeutic effect.
Thus at low doses, MPA disrupts the production of GnRH, thechemical signal sent out by the brain to sex glands. Athigher doses it is effective in the sex glands themselves.
"This knowledge gives us some new tools in working withthese diseases," Auchus said. "For example, there are otherdrugs that people use in gonadotropin independent precocity,and we now know that they work by inhibiting different partsof the hormone-producing pathway than MPA does. That maymean that additive therapy would be effective severaldrugs working on different pathways, each at lower dosesthan if it were used alone."
In addition, Auchus said, now that they have shown thatinhibiting this one enzyme can influence sex hormoneproduction, 3bHSDII may turn out to be a good target enzymeto enhance treatments of breast cancer.
YEAST CELLS AS LITTLE ADRENAL GLANDS
Lee's and Auchus' discovery actually was a small part of a much larger project, undertaken in the Miller lab under Auchus'initiative. The group has developed a strain of "humanized"yeast that express the DNA of human enzymes. They plan touse yeast cells, converted into little versions of humanadrenal glands, as a scientific model to study the action ofthe human enzymes that are involved in sex steroid hormoneproduction
Lee worked in the Miller lab on this project during ayear-long stint at UCSF, between his graduation from UCBerkeley and matriculation as a Tufts University medicalstudent. The MPA project is one of several to use the newyeast strains.
With their new model, for the first time the scientists canstudy the biochemical action of a single enzyme. Also, theycan study the actions of drugs on each enzyme needed forhuman steroid production.
"All human cells handle cholesterol, whereas yeast containsno cholesterol," Auchus explained. "The value of using yeastcells, instead of human cells, for these studies is that wecan put in known amounts of the enzyme, the cholesterol andall the proteins the enzyme has to interact with, to workout the individual biochemical steps in the enzyme's action.
"With this system we can ask questions about how humanadrenal glands and gonads make hormones," Auchus said. "Wedon't know, for example, how a cell in the testes or theovaries decides to make one steroid as opposed to another.The yeast system will permit us for the first time to workthis out with rigor."
Auchus, Miller and Lee first used "humanized" yeast to studythe biochemistry of the key cholesterol-converting enzymecytochrome P450c17. The group's paper on the biochemicalactivity of cytochrome P450c17 was published in February,1998 in the Journal of Biological Chemistry.
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