Porphyrins, largely neglected by biotechnology and pharmaceuticalfirms alike, are coming to the fore in California and Texas.A "rapid communication" paper in the Aug. 10 Journal of theAmerican Chemical Society (JACS) reports site-specific cleavage of anRNA molecule by a synthetic, conjugated, porphyrin-mimickinganalog _ texaphyrin.Pharmacyclics, Inc., of Sunnyvale, Calif., performed the experiment,which, its authors wrote, "augurs well for the use of such conjugates inantisense applications," as cancer, hepatitis and atherosclerosis.Porphyrin, a ubiquitous molecule that binds various metals, occurs invirtually all cells. Heme, for example, the oxygen-carrying, rust-colored, iron-chelating component of hemoglobin, is a porphyrin. So iscobalt-embracing vitamin B12. So is chlorophil, the magnesium-binding agent of photosynthesis, by which green plants convert visiblelight photons into chemical energy.Texaphyrin, as its name hints, is a large-size, lone-star-shaped analogof porphyrin, created by chemist Jonathan Sessler at the University ofTexas in Austin. Sessler is a co-founder of Pharmacyclics, and a co-author of the JACS paper. So is oncologist Richard Miller, thecompany's president and chief executive officer."Texaphyrin," Miller told BioWorld Today, "is a synthetic, small-molecule, non-recombinant porphyrin, with some built-in features thatenable it to bind particularly interesting metals." First and foremost, sofar, the rare-earth metal, europium."Rare-earth metals," Miller explained, "are not so rare, actually. Theycomprise 1 percent of the earth's crust." Europium, with atomicnumber 63, is just over half-way along the periodic table of theelements, between hydrogen (number 1) and lawrencium (number103). Though fairly rare, Miller said, europium is not precious; itranges in price "somewhere between calcium and gold."But this metal's price is above rubies, in the part it plays upgradingporphyrin to texaphyrin. "Nature's porphyrin," Miller explained, "hasfour nitrogens, which form a pocket. Texaphyrin has five."Expanding Porphyrin Star From Four Points To FiveThat fifth nitrogen, he explained, "gives you more room, a biggercavity, allowing one to put in larger metals." Pharmacyclics synthesizestexaphyrin "from pretty simple starting materials, such as acetone andother solvents, by a several-step organic-chemistry process."Miller added, "When Sessler, its inventor, looked at the X-raycrystalographic structure of the molecule, with its five nitrogenouspoints, looking like the Star of Texas, he thought it would be prettyneat to name it texaphyrin. And that name has stuck in the literature,including some nine or 10 issued U.S. patents."Miller compares the texaphyrin molecule to a doughnut, in which thefive nitrogen atoms face inward toward the hole, there chelating theeuropium. They do so in a special way, "tightly binding the metal atomaround its equator, like the belt around your waist, leaving its north andsouth poles free to interact with other molecules."In the just-published proof-of-concept paper, those moleculescovalently conjugated to the metal were short _ 20-unit polymer _synthetic DNA sequences of a gene that encodes p glycoprotein, amultiple-drug-resistance (MDR) protein expressed in tumors. TheseDNA strands were complementary to the RNA generated to synthesizethe drug resistance factor, and were designed to chop it off and chew itup by site-specific hydrolytic cleavage.In such a catalytic process, he emphasized, "the Eu-Tex conjugate notonly binds to the target RNA, but also cuts its template to pieces. Soone molecule of construct can destroy many copies of RNA.""We used the MDR gene," Miller explained, "because we're interestedin oncology applications, and as a demo model. The p glycoprotein onthe surface of cancer cells is responsible for drug export." He observed,"Nature is smart and has this protein that pumps things out of the cell,among which are chemotherapy drugs. One way tumor cells becomeresistant to such drugs is by making more messenger RNA, and soamplifying that gene."The europium ion has a very high affinity for the oxygen in thephosphates of the nucleic-acid backbone. When the texaphyrin "sees"these, it pulls all of their electrons off, allowing a water molecule, H20,to insert and cleave.This is art imitating nature, Miller observed. "Nature uses the samechemical reaction that we've used." Specifically, after RNA loops outthe non-coding introns from its sequence, prior to transcribing a newprotein, a part of that intron, which is a ribozyme, hydrolyzes, and socleaves, the adjoining exon sites."So we tried to make an antisense ribozyme analog, or mimic, namely,europium-texaphyrin."Helping Antisense To Make More SenseMiller thinks that the road being traveled by companies and researcherspursuing antisense technology "has become tortuous, with dauntingquestions and potential limitations." He explained:"When people thought about antisense as drugs, there was the usualeuphoria: `What a clever idea.' Then came coming to grips with reality_ it's got to get into a cell; it's got to be stable; it's got to get to theright parts of the body."He quickly added, "There's a lot of smart people working on antisense,and little by little solving these problems. Here's how texaphyrins canhelp potentially to overcome the present limitations of antisense:u "They get into cells very well, cancer and liver cells for example. Sohaving a texaphyrin on an antisense sequence would facilitate cellentry.u "Antisense binds to messenger RNA. To which we say, `So what?Your cell can continue to make more mRNA, and crank out moreprotein. So if you just tie up a few of them, you may not have reallyachieved very much.' Texaphyrin, by its ability to cut and then go onto cut again, offers potentially increased messenger destruction."Right now, Miller and his colleagues are conjugating europium-texaphyrin to an oncogene. "We're going to start to move into animals,and see if we can have a role in anti-tumor systems." Because theconstruct goes preferentially to liver cells, they will also try it forcontrolling hepatitis C.But europium, Miller emphasizes, is not the only element inPharmacyclics' palette of rare-earth metals. Later this month, a PhaseIII trial will test gadolinium (atomic number 64) as an oral contrastagent for magnetic resonance imaging of the gastrointestinal tract. Andbecause of its attraction for cholesterol, athersclerotic plaques are alsoon their hit list. Gadolinium has seven unpaired electrons, more thanany other element.Lutetium (no. 71) has no unpaired electrons, so _ like chlorophil _photosynthesizes light energy into chemical energy. This property haspotential for enhancing the X-ray or other ionizing radiation used incancer therapy. n
-- David N. Leff Science Editor
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