By David N. Leff

Science Editor

A humble soil fungus named Trichoderma polysporum transformed organ transplantation from the dicey operation of last resort it was 20 years ago into the standard surgical procedure it is today.

Looking for new antibiotics circa 1970, medicinal chemists from Sandoz Pharma AG, of Basel, Switzerland, isolated the fungal organism in soil dug up in Norway. A decade later, one of T. polysporum's metabolites, dubbed cyclosporin A, was found to fight not pathogens but the human immune system.

This new drug proved far superior to the steroid immunosuppressants then in use to prevent a transplant recipient's immune defenses from rejecting his or her new but alien organ graft. However -- and it's a big "however" -- cyclosporin A brings with it a cruel paradox: Its toxic side effects often damage severely the very organ it has rescued from rejection.

Recent data show that the drug damages 25 percent of implanted donor kidneys, 38 percent of new hearts, and 37 percent of livers. Its other side effects range from impairing the nervous system, increasing blood pressure and causing malignant lymphomas, to skin disorders, notably socially disfiguring facial hair growth. (An ironic aside to this last cosmetic stigma is the fact that that fungus's name, Trichoderma, means "hairy skin" in Greek. )

When a transplanted organ -- except one from an identical twin -- takes up residence in its new body, the recipient's immune system launches a fast, ferocious attack on what it sees as a huge new kind of lethal infection. Killer T cells lead the charge, and unless protected by timely immunosuppression, the gift graft is abruptly rejected, in minutes or hours.

The prime mover in this search-and-destroy operation, explained molecular biologist Patrick Hogan, "is a receptor on the T-cell surface, which recognizes the strange antigen, together with an MHC major histocompatibility (MHC) protein. Other immune-system arms, such as antibody-forming B cells and phagocytic macrophages, also make cytokines in response to receptor engagement."

Hogan proposes that "T cells have a critical role in turning up the immune response, so they may be the first target for cyclosporin A." His laboratory in the Harvard-affiliated Center for Blood Research focuses on developing new immunosuppressive molecules, free of the current drug's side effects.

A key player in this pathway from T cell receptor to immune reaction is calcineurin, an enzyme that transmits signals from that receptor to the cell's nucleus. It acts, Hogan explained, "by taking phosphate groups off a variety of proteins."

"In fact, " he added, "calcineurin is expressed not just in the immune system, but in the brain, the liver and everywhere else in the body."

In the immune system, an important target for calcineurin is a family of transcription factors called "nuclear factor of activated T cells, " or NFAT for short. "They are probably the main downstream proteins that are inhibited by cyclosporin A, " Hogan surmised.

"Now, when these NFAT proteins are turned on by the action of calcineurin, " he went on, "they go from the T cell's cytoplasm into its nucleus. It all happens very fast: the receptor is engaged on the cell surface; calcineurin gets activated and dephosphorylates NFAT proteins, all in about a minute."

Hogan is senior author of a paper in the current issue of Molecular Cell (a spin-off of the journal Cell), dated April 24, 1998. Its title: "Selective inhibition of NFAT activation by a peptide spanning the calcineurin targeting site of NFAT."

That peptide is the Harvard group's entry in the sweepstakes to someday supplant cyclosporin A with a less damaging immunosuppressant. So far, it's in the in vitro cell-culture lane of that race track.

The co-authors prepared a panel of NFAT proteins with alanine amino acids substituted at 58 positions. Three of these mutants, they report, "showed a striking inability to translocate to the nucleus" of the cell.

"We call this candidate molecule, " Hogan recounted, "the SPRIEIT peptide, based on its critical amino-acid sequence in NFAT -- using the one-letter amino-acid code."

SPRIEIT differs from cyclosporin A, Hogan pointed out, "in the region on calcineurin to which each of them binds. Cyclosporin A blocks calcineurin's active site, so it can't act on any other molecules in the cell. SPRIEIT on the other hand, " he continued, "blocks only specific binding between calcineurin and NFAT, leaving its other functions unimpeded."

Hurdles En Route From Peptide To Pill

Peptides themselves, Hogan observed, "are not drug candidates; they are too hard to deliver. But what our STRIEIT peptide work shows is that one can interfere with the immune-system pathway, using a small peptide.

"So now it becomes a new target point for us to get in with a small molecule, and see if we can develop a more promising immunosuppressant drug lead: something that's not a peptide, that can be delivered orally, and will attack the same sites a peptide does, without the side effects of cyclosporin A."

Before "eventually" moving into in vivo lab-animal experiments, Hogan and his co-authors, he said, "have to get over this hurdle of being able to deliver a drug into cells. We did that in cells in culture by the trick of making a cDNA protein fusion that will carry them in. But that's not really a very promising way of doing efficacy or toxicity studies in animals."

Harvard's Center for Blood Research, Hogan said, "has filed a patent application on the STRIEIT peptide and on the assays that would be used to go from peptides to other molecules that bind to the same site, and would be processed in the same way."

Because calcineurin and NFAT are so widespread in the body, Hogan foresees extending their effects beyond immunosuppression. A drug or drugs based on their work in progress, he suggested, "could be used to treat chronic conditions caused by excessive or inappropriate immune responses -- such as asthma, inflammation, allergies and rheumatoid arthritis. *