While debate rages on over free exchange of drug addicts' needles to prevent the spread of AIDS, the biotechnology and pharmaceutical industries are pursuing a far broader needle-exchange agenda.
"From a pharmaceutical point of view," molecular biologist Jonathan Rosen told BioWorld Today, "we are interested in finding small molecules that mimic large-size proteins and peptides, such as growth factors and cytokine hormones. Compounds that can be taken in pill form rather than by injection."
Rosen is senior director of transcription research at Ligand Pharmaceuticals Inc., in San Diego, Calif., and senior author of a paper in the current issue of Science, dated July 10, 1998. Its title is "A small, nonpeptidyl mimic of granulocyte-colony stimulating factor [G-CSF]."
G-CSF is a billion-dollar-a-year injectable drug developed by Amgen Inc., of Thousand Oaks, Calif., and sold worldwide under the trade name Neupogen.
Amgen manufactures recombinant Neupogen in Escherichia coli host cells. It's a cytokine hormone that reverses the disastrous decline of immune-system neutrophils in severe infections. Neutrophils make up from one-half to two-thirds of the blood's infection-fighting white cells.
Neupogen's prime use is fighting off the infections that arise in cancer patients treated with chemotherapy that dangerously drops their neutrophil counts. It's also prescribed for bone-marrow transplantation and advanced opportunistic infections in AIDS patients.
Ligand and its partner, SmithKline Beecham plc (SB), of London have drawn a bead on G-CSF as a likely first target of their aim to replace large proteins with mini-mimic molecules.
"The ultimate goal of our joint program," Rosen said, "is to find a small molecule that mimics G-CSF and can be taken in pill form rather than injection as the parent protein is."
Their point of departure was a small synthetic molecule turned up by high-throughput screening of SB's defined compound collection, and numbered SB 247464. "It was initially chosen," Rosen recalled, "based on its ability to activate the G-CSF receptor."
Like other cytokines — which the immune system dispatches to the scene of infection — G-CSF works by galvanizing that portion of its receptor protruding from its cell surface. It takes a sizeable protein to touch off this activation. If swallowed, the stomach acids would degrade it, so it must be injected. A much smaller, orally available molecule could presumably evade this barrier and limit unwanted side effects.
Ligand's lightweight SB 247464 compound successfully gave the heads up to murine G-CSF in vivo, in mice.
"I think from a scientific point of view," Rosen observed, "it was quite a surprise to some people that it was possible to do this at all. What we've been able to mimic is essentially the interaction of one reasonably large protein with a protein receptor. Instead of using G-CSF, which is a protein interacting with its natural receptor, we've done it with a small, unrelated molecule, SB 247464."
How small is small?
In the way a boat is smaller than a ship, peptides are smaller than proteins. Ligand's nonpeptidylic compound is an order of magnitude smaller yet. "SB 247464," Rosen pointed out, "is a symmetrical, heterocyclic, organic compound with a molecular weight of 526 daltons."
He recalled the case of erythropoietin (EPO), developed by Amgen in 1989. Originally approved to treat the anemia of kidney dialysis, recombinant EPO (trade named Epogen) also is prescribed against the blood-degrading side-effects of AZT in treating HIV infection.
In the year of Epogen's discovery, Johnson & Johnson Research Institute, in Raritan, N.J., enlisted the Affymax Research Institute, in Palo Alto, Calif., to help find a smaller molecule that did the same job as EPO.
Two years ago, they came up with a dimeric (double-molecule) peptide weighing in at close to 5,000 daltons vs. EPO's 34,000. (See BioWorld Today, July 26, 1996.) "That's ten times the mass of our small molecule," Rosen pointed out. Neupogen weighs 18,000 daltons, with so far no peptide version in sight.
"Having a peptide isn't really that much of an advance over having a protein," Rosen commented. "It's still going to get degraded in the stomach."
Ligand molecular biologist Peter Lamb, principal author of the Science paper, explained why cytokine hormone receptors have to double up to go to work.
"Normally, when G-CSF or other cytokines of that type interact with cells, they bind to their receptors. The first key event is that this causes receptor oligomerization, which is just a clustering of receptor chains," he said.
Receptor Pathway Requires Two-Passenger Twinning
"That clustering," Lamb went on, "is the on switch, if you like, that initiates the signal transduction process inside the cell and ultimately leads to changes in cell behavior. So we think that our small-molecule compound also causes this receptor clustering effect. And that's how it's able to mimic the G-CSF effect."
In mice, yes; in humans, no. Natural G-CSF is species non-specific; not so, the synthetic, murine version.
"Although this compound does have, we believe, incredible effects in the mouse," Rosen observed, "in the assays we've done to date we have not seen activity in human systems. So we're going back to several drawing boards, looking at related molecules from the same series as well as discovery of additional ones that will be human-active."
Aside from what he termed "that small caveat," Rosen pointed out, "There are a number of ways to go with this particular product. One is we'd like to understand in more detail how exactly the mimicking effect's working. We have some thoughts on that, but we'd like to proceed and see if they're correct.
"Clearly too," he continued, "we'd like to do more screening and try to find additional compounds that have this kind of effect. Finally, we're looking at a series of variants of SB 247464 to see what kind of activities they may have."
Rosen concluded, "This is a general approach to drug discovery that's applicable to many protein receptor systems. It's an important lesson that we learned — and I'm afraid many others will learn from our work." *