Conclusions Selex Lessons for Drug Discovery

Aptamers are discovered through a combinatorial chemistry paradigm. At the bench, with something like 1015 potential single-stranded DNA or RNA aptamers in a test tube, various target molecules are used to screen the library to find ap-tamers that bind most tightly and selectively. The paradigm is decidedly not a rational drug design method. Within the large numbers of molecules in the library one asserts that one or more sequences fold into an "attractive" globular shape for binding with high affinity and specificity to the target. No attempt has been made (in spite of much pressure to do so) to "design" the oligonucleotide pools to be target "friendly" - when that was tried, nothing profound happened.

Craig Tuerk and I understood one big thing, which was that the intricate three-dimensional shapes of single-stranded oligonucleotides, including the vast structural possibilities within the unpaired nucleotides, were sufficient to think of aptamers as nucleic acid antibodies. However, one might like to do SELEX with oligonucleotide libraries that include better chemistry; that has been achieved through the beautiful pyrimidine chemistry of Bruce Eaton (Eaton, 1997). One might imagine that the future of SELEX will include five position-modified pyri-midines such that the oligonucleotides can still be amplified (and thus SELEXed) but that the resulting chemistries will be even more "protein-like." In fact, this is a large part of what we do at SomaLogic.

SELEX is not typing; aptamers must be identified at the bench, even though the SELEX paradigm can be routine. There are drug discovery paradigms that are typing - antisense, ribozymes, and now siRNAs. For a long time people have hoped that a "typing" paradigm would work, and the latest candidate technology - siRNAs - is now in its euphoric stage. In the final analysis typing-based oligo-nucleotides (and aptamers as well) will struggle with the cost of goods, with aptamers (aimed at extracellular targets, sensibly) having an advantage over those molecules that must enter a cell to function (such as the "typed" drugs, which are always aimed at oligonucleotide targets). Aptamer therapeutics will succeed or fail more because of the cost of oligonucleotide synthesis (which will come down over time) than because of the intrinsic affinity or selectivity of aptamers - aptamers have wonderful affinity and specificity.

More interestingly, SELEX and aptamers may have something profound to say about combinatorial chemistry methods aimed at finding orally active small molecules. Combinatorial chemistry is not in as much favor today as it once was, probably because no one has made large enough libraries to screen, and no one has figured out how to screen efficiently. Can huge libraries, well screened, yield better orally active compounds than we are used to seeing? (After all, apta-mers compete well with antibodies, and in many cases have higher affinities and specificities - is that because SELEX uses huge libraries or because oligonucleotides have better chemistries than proteins? Probably aptamers with terrific properties are identified because of the starting library size and not the functional advantages of RNA or DNA over proteins; as Bruce Eaton once noted (personal communication), RNA and DNA chemists should have "side group" envy toward the amino acids.)

There is a lesson for finding small, classic, orally active drugs. One might select orally active compounds from enormous libraries or one might quickly identify some weak "hit" present in smallish libraries and ask the medicinal chemists to develop nanomolar and picomolar compounds based on those indifferent compounds. Today's chemists work with focused libraries that are built from weak hits, largely because no one can change the game by giving the chemists better hits as their starting materials.

SELEX provides both high-quality compounds (with picomolar Kj-values) and a useful paradigm for finding better small molecules as hits. The dual requirements for terrific drugs are high affinity and specificity, which can be obtained even with the limited surface interactions available from a compound with a molecular weight around 700 or so. It is not axiomatic that small molecules must have low affinity and specificity (and thus unexpected side effect profiles from non-target interactions) - we can look at biotin binding to avidin and think that our screening protocols are more limiting than is chemistry itself. The available shape space for medicinal chemists is adequate for better drugs than we are used to seeing. While SELEX may be a source of strong aptamer therapeutics for many years to come, SELEX also has taught us that large libraries have better winners than smaller libraries, a deep and obvious lesson from the SELEX experience of the last 15 years.

468 | A Personal Perspective: Aptamers after 15 Years Acknowledgments

SELEX could not have happened without Craig Tuerk. Nebojsa Janjic is the person who facilitated, more than anyone else (even as others contributed), the proof that aptamers could be drugs. Aptamer development on a broad scale was a function of many friends and colleagues at NeXstar and more recently at SomaLogic, Archemix, NOXXON, and Eyetech.

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