The small-molecule approach to biology

Tags: small molecules, chemical genetics, chemical space, approach, information transfer, reverse genetics, Harvard Medical School, organic chemistry, genetic approaches, genetic screens, ChemBank, Stuart L. Schreiber, signaling protein, modern biology, complex networks, Philip Leder, target, organic chemist, synthetic organic chemist, Cell signaling, Caroline Shamu, biological assays, ICCB codirector Timothy J. Mitchison, organic computer, mitochondrial function, Howard Hughes Medical Institute, Harvard mesenchymal progenitor cells, Exploring biology, Harvard University, Stanley J. Korsmeyer, Solomon H. Snyder, binding protein, mechanistic studies, classical genetics, signaling pathways, Signaling networks, natural products, organic synthesis, divergent synthesis, chemical biology, generality, Takeda Chemical Industries, Stanford University, type 2 diabetes, C. David Allis, Vincent G. Allfrey, Johns Hopkins University, somatostatin receptor, Rockefeller University, molecular cell biology, Gerald R. Crabtree, organic chemists
GENERATING DIVERSITY Using split-and-pool synthesis, a small-molecule intermediate is split into numerous reaction vessels prior to a subsequent step in a divergent synthesis pathway. The process yields complex and skeletally diverse small molecules, which can be screened for biological activity. THE SMALL-MOLECULE APPROACH TO BIOLOGY Chemical genetics and diversity-oriented organic synthesis make possible the systematic exploration of biology
STUART L. SCHREIBER, Harvard University S mall molecules have long been associated with biological discoveries, but, in contrast to biochemical and genetic approaches, the small-molecule approach has lacked generality. Although the advances made through the use of small molecules as probes (as distinct from medicines) are impressive, they have in general come about on a case-by-case basis. Advances in diversity-oriented organic synthesis and a focus on the underly-
ing similarities between the small-molecule and genetic approaches have increased the generality of the small-molecule approach. Although a truly systematic way to explore any and all facets of biology with small-molecule modulators has not yet been reached, these advances are beginning to influence researchers on a much broader scale. It is becoming more common for a life scientist to ask, "Should I tackle this problem with small molecules?" or even to state, "The only way I can tackle this problem is with small molecules."1 The latter is becoming increasingly com-
C&EN / MARCH 3, 2003 51
mon as global views of biology are sought. The discovery principles and platforms enabling this transformation constitute what Rebecca Ward at Harvard University first coined the "chemical genetic" approach on the cover of the inaugural issue of Chemistry & Biology nine years ago. Her term reminds us that, to understand a life process, you should perturb it and determine the consequence and that such an approach should strive to have the broad generality and power of genetics. That is, it should allow the probing of life processes in both a systematic and thorough way (analogous to the application of genetics and to the use of saturation mutagenesis, respectively). Chemical genetics is a logical outgrowth and subset of chemical biology, where chemical principles and techniques are used to dissect directly, rather than to model, biology. In this perspective, I aim to focus on the current transition from the ad hoc to systematic use of small molecules to explore the life sciences (rather than to discover new medicines) and the role of Organic Chemistry in mediating this transition. The same experiments that encouraged me to explore biology with organic chemistry frustrated me as well. These frustrations had to do with, at the time, the inability of the small-molecule approach (in this context, sometimes referred to as the "pharmacological approach") to be applied with the broad generality of the reductionist-based biochemical and discoverybased genetic approaches. As a result, I was left with an uneasy feeling about the role of organic chemistry in future studies.Asecure role for organic chemistry would entail its use "front and center" as a general discovery engine, rather than relying on chance opportunities provided by neighboring disciplines. Overcoming these frustrations required adapting the principles that underlie genetics (and more recently genomics) to chemistry--a process that is proving to be a fertile one for chemistry. For example, much as natural products have driven the development of both target-oriented synthesis (TOS) and synthetic methods, chemical genetics is providing a driving force for the development of diversity-oriented synthesis (DOS; see below). From a century of genetics-based interrogations of life, we have learned that perturbing life processes and observing the consequences can provide illuminating insights. Geneticists do so through the use
of gene mutations, either naturally occurring, randomly induced, or targeted. They have developed powerful analysis tools, such as "epistasis analysis" to order genes in pathways, "synthetic lethal screening" to reveal redundant elements of pathways and networks, and "modifier (suppressor and enhancer) screening" to reveal connections between pathways and networks. These principles and analysis tools are directly applicable to chemical genetics. Here, of course, the wild-type protein is used; it is the binding of a small molecule to the protein that results in a perturbation of function, either inhibition or activation. Recognizing this parallel alone, however, does not ensure a general approach to exploring biology.Aresearch infrastructure involving new advances in chemistry must be developed and integrated into the fabric of day-to-day life science research. It must be routine and readily available to life science researchers, especially to chemists and biologists. In the sections of this perspective, I describe the use of small molecules that have illuminated life processes, the shortcomings of this type of research as a general approach, efforts to overcome these shortcomings, and an assessment of where we stand today. AD HOC USE OF SMALL MOLECULES TO EXPLORE BIOLOGY.The past century has yielded many examples of researchers identifying and using small molecules to probe aspects of biology. Some of these served as the subject of this article's prequel, authored 10 years earlier.2 Cytoskeleton. GaryG.BorisyandEdwin W. Taylor's use of colchicine, at the MRC Laboratory of Molecular Biology, in Cambridge, England, to identify the tubulin proteins is a classic illustration of using small molecules for discovery in basic biology.3 Colchicine, along with numerous recently discovered small molecules, targets the -tubulin/-tubulin protein-protein interface of microtubules and thus disrupts microtubules in cells. Despite the widely held view that small molecules generally fail to disrupt protein-protein interactions, this interaction appears to be a particularly simple one. For example, in one small-molecule screen, over 300 of 16,000 small molecules were shown to have this property, while two were found to be stabilizers of the same protein-protein interaction.4 Such molecules have illuminated the functions of microtubules as key cytoskeletal elements.
Cytoskeletal research in general has been a rich beneficiary of small-molecule probes.3b Microtubule-stabilizing agents such as paclitaxel (Taxol) played an important role in the identification of microtubule-associated proteins (MAPs). Actindisrupting agents such as cytochalasin and latrunculin have played key roles in unraveling the mysteries of the actin cytoskeleton, and recently discovered small molecules that specifically target motor proteins that carry their cargo along the actin and microtubule polymers are now being used to reveal previously hidden facets of motor function.3b Recent experiments concerning the regulation of the cytoskeleton suggest that the use of small molecules to understand this area of research will continue in the future. Ion channels and signaling in the neurosciences. Neurobiology has long been a beneficiary of the ability of small molecules to target the neurotransmitter receptors and ion channels that function in neurons. As a result, neurobiologists tend to be among the most eager to see advances in chemistry relevant to small molecules. Natural products, especially from snake, spider, bee, scorpion, dinoflagellate, pepper, snail, puffer fish, and soft coral, have played a particularly prominent role in these studies.5 Scientists at Pfizer, recognizing the treasure trove of ion channel probes stemming historically from spiders, recently developed a fruitful effort in both the isolation of channel-blocking natural products from spiders and the synthesis of optimized variants. These small molecules were used to classify channel subtypes and to probe their functions in neurobiology. At the National Institutes of Health, Arvid Carlsson's use of reserpine, L-DOPA, and chlorpromazine led to the discovery of the neurotransmitter dopamine and to its role in mediating signals within the nervous system ("chemical transmission in the brain").6 His studies of dopamine and the dopamine receptor, and of antagonists of their interaction such as chlorpromazine, provided early hints of how extracellular factors, without entering a cell, can give rise to changes in intracellular processes-- in other words, signal transduction. For these discoveries, Carlsson was awarded a share of the Nobel Prize in Physiology or Medicine in 2000. Inner leaflet of the plasma mem- brane. Studies of the phorbol diesters
"There should be no problem with biology driving science unless perhaps you happen to be a chemist!"
52 C&EN / MARCH 3, 2003
Somatostatin receptor agonist
Phorbol 506BD
Dimerizer (bumped rapamycin)
K-trap affinity reagent
SMALL-MOLECULE PROBES Colchicine, a probe of tubulin3; spidamine, used to study glutamate receptor function5; reserpine, used to discover the neurotransmitter dopamine6; phorbol (parent alcohol of a family of diesters), used to study a family of protein kinases7; pioglitazone (Actos), an activator of the transcription factor PPAR8; MK-886, used to discover the protein 5-lipoxygenase-activating protein (FLAP)9; somatostatin receptor agonist, used to study a specific receptor's Physiological Functions10; 506BD, a probe of immunophilin action11,12; dimerizer (methallylrapamycin), an inactive ("bumped") variant of rapamycin that, by Chemical Modification, gained the ability to control proximal relations of signaling proteins in cells and animals17; rapamycin, a probe of the nutrient-response signaling network and of the proteins FRAP and TOR20; K-trap affinity reagent (lysine variant of trapoxin), used to discover HDAC1.23
played an important role in revealing the key functions of members of a large family of protein kinases named "PKCs" in intracellular signal transduction.7 They also revealed a docking site used by these proteins to associate with the inner leaflet of the plasma membrane. Phorbol diesters bind to the PKCs and tether them to the leaflet, thereby creating proximal relationships with their leaflet-localized substrates. These studies demonstrate how small molecules can activate the functions of the proteins to which they bind by directing them to specific locales within cells. Biological insights stemming from pharmaceutical research. Researchers in the pharmaceutical and biotechnology industries, while in search of new medicines, have been particularly effective at discovering new insights into life process-
es using small-molecule probes. As with all studies using small molecules, two approaches have been used: One emulates the underlying principles of classical genetics (sometimes called "forward genetics") and the other a modern variant of it, reverse genetics. As an illustration of forward chemical genetics, small molecules were screened at many companies in the 1970s in search of agents capable of treating type 2 diabetes. Pioglitazone, the archetype of the "glitazones," was discovered at Takeda Chemical Industries, in Japan, during this period. Only in more recent years has the target of the glitazones been determined. In accordance with insights gained from human genetics, the glitazones bind and activate the nuclear receptor PPAR.8 It is now known that PPAR plays a key role in di-
abetes, although that role is still mysterious, as is the molecular etiology of the disease. A second example of this approach derived from research aimed at understanding the molecular basis of inflammation. In mechanistic studies of the anti-inflammatory agent MK-886, this small molecule was used to discover 5-lipoxygenase-activating protein (FLAP) and to assign its cellular and physiological functions.9 These studies opened the door to a new area of research involving FLAP and its role in inflammation. As an illustration of reverse chemical genetics, small molecules have also been identified that selectively bind and activate five members (paralogs) of the somatostatin receptor family. Having predetermined the selectivity of these probes toward individual paralogs, scientists at
C&EN / MARCH 3, 2003 53
Merck were able to uncover the distinctive functions of the paralogs.10 This remarkable investigation used the logic of reverse genetics, in that the researchers targeted the modulation of function (in this case, the more challenging activation of function) of individual paralogs, then searched broadly for the resulting consequences. Signaling networks in the cytoplasm and nucleus. In my laboratory in the early and mid-1980s, a focus on target-oriented synthesis and on developing synthetic methods eventually led to studies of how the small-molecule objects of our studies perturb the functions of the proteins to which they bind. These studies using natural products allowed us to discover new principles of biological signaling networks, including the commonality of principles underlying both cytoplasmic and nuclear signaling networks. Four studies of natural products revealed basic insights into information transfer in biology. The discovery of the FK506-binding protein FKBP12 in 1988 (independently discovered in my lab and by scientists at Merck)11 was facilitated by target-oriented synthesis efforts aimed at, among others, the natural products FK506 and rapamycin and the nonnatural small molecules 506BD and tricyclosporin.12 Using a number of these small-molecule probes, we determined in 1991 that FK506 and cyclosporin inhibit the activity of the phosphatase calcineurin. This occurs by an unusual mechanism: through the formation of the ternary complexes FKBP12FK506-calcineurin and cyclophilin-cyclosporin-calcineurin.13 This work, together with work by Gerald R. Crabtree at Stanford University concerning the NFAT proteins, led to the elucidation of the calcium-calcineurin-NFAT signaling pathway.14 This proved to be an early example of defining an entire cellular signaling pathway from the cell surface to the nucleus, analogous to that of the Ras-Raf-MAPK pathway elucidated the following year. In subsequent years, the central roles of the calcium-calcineurin-NFATsignaling pathway in immune function, heart development, and memory acquisition were revealed by many researchers working in a variety of fields.15 The ability of small molecules to bind two proteins simultaneously inspired our collaboration with Crabtree and members of his laboratory in 1993 to develop "smallmolecule dimerizers," which were shown to provide small-molecule regulation of transcription and of numerous signaling molecules and pathways (for example, the Fas, insulin, TGF, and T-cell receptors16, 17)
through proximity effects. We demonstrated that small molecules could be used to influence signaling pathways in an animal with temporal and spatial control.18 Subsequently, many researchers working on many research problems have had success with this approach, and dimerizer kits have now been distributed freely to more than 500 laboratories by Ariad Pharmaceuticals.19 Its promise in gene therapy has been highlighted by the stable (over several years), small-molecule-induced production of erythropoeitin (EPO) in primates by treating primates with a small-molecule switch for an EPO-inducing transcription factor, and more recently, in Phase II human clinical trials for treatment of graftversus-host disease. Members of my group and of Solomon H. Snyder's group at Johns Hopkins University independently discovered in 1994 that the small molecule rapamycin simultaneously binds FKBP12 and the previously unknown protein we named FRAP (FKBP12-rapamycin binding protein, also known asTORand RAFT).20 Using chemical genetic epistasis analysis21, diversityoriented synthesis, and small-molecule microarrays, among other techniques, we succeeded in uncovering the nutrient-response signaling network involving TOR proteins in yeast and FRAP/TORin mammalian cells. Small molecules such as uretupamine22 and rapamycin were shown to be particularly effective in illuminating the ability of proteins such as FRAP, Tor1p, Tor2p, and Ure2p to receive multiple inputs and to process them appropriately toward multiple outputs ("multichannel processors"). The nutrient signaling network now appears to be key to understanding the origins of type 2 diabetes. In 1996, my lab used a synthetic variant (an immobilized version of K-trap, page 53) of the natural product trapoxin to molecularly characterize the histone deacetylases (HDACs).23 Prior to our work in this area, the HDAC proteins had not been isolated--despite many attempts by others who were inspired by Vincent G. Allfrey's detection, at Rockefeller University, of the enzymatic activity in cell extracts more than 30 years earlier. Coincident with the HDAC discovery, C. David Allis and colleagues at the University of Rochester, taking a biochemical approach, reported their discovery of the histone acetyl transferases (HATs).24These two contributions catalyzed much research in this area, eventually leading to the characterization of numerous histone-modifying enzymes, their resulting histone "marks," and numerous proteins that bind to these marks.
By taking a global approach to understanding chromatin function, we recently proposed a "signaling network model" of chromatin and compared it with an alternative view, the "histone code hypothesis" presented by Allis.25 Research by many scientists in this area has shined a bright light on chromatin as a key regulatory element, rather than simply a structural element, in transcription. This research followed previous small-molecule-based studies of signal transduction that helped reveal the existence of cytoplasmic signaling networks. The signaling network model of chromatin25 posits that both cytoplasmic signaling and chromatin signaling use key elements of networks, including feedback motifs and redundancy that ensure robustness, adaptability, and switchlike behavior. This insight was gained in part by recognizing the remarkable similarities in the principles that underlie information transfer in the cytoplasm and nucleus (page 55). Our view of information transfer in cells has been extended from the early events of signal detection, often at the plasma membrane, to chromatin, where memory of the signal is established in nondividing cells, and inheritance of the signal (epigenetics) is achieved in dividing cells. The concept of signaling pathways (in my opinion, an artifact of the reduction approach) has been evolving toward the concept of signaling networks. The fine temporal control of protein function in cells afforded by small molecules has played a key role in this transition.25 SYSTEMATIC USE OF SMALL MOLE- CULES TO EXPLORE BIOLOGY: CHEMI- CAL GENETICS. From the perspective of an organic chemist participating in the study of small-molecule-based signaling networks in 1997, I could not help but be concerned with two issues. First, the skill set of an organic chemist was well suited for responding to discovery opportunities provided by biologists, but not necessarily for initiating or leading the discovery program. This is, in retrospect, not surprising, as it mirrors the role of organic chemists in the process of modern drug discovery. In contrast to drug discovery prior to the mid-1970s, with the advent of molecular biology and molecular cell biology, organic chemists are typically asked to participate in the optimization process following the decision by biologists to select a specific biological target for therapeutic intervention. This concern is admittedly a selfish one--there should be no problem with biology driving science unless perhaps you happen to be a chemist!
54 C&EN / MARCH 3, 2003
based investigations of membrane-to-nucleus
signaling and chromatin function suggest the
existence of network motifs that ensure robust-
ness, adaptability, and switchlike behavior, as
Kinase subdomains
well as other striking commonalities (the signaling network model of chromatin).25 The figure
uses the platelet-derived growth factor receptor
(PDGFR) and a nucleosome to illustrate that
Kinase Tail insert Extracellular signal leads to receptor dimerization, trans-phosphorylation
Nuclear signal leads to lysine acetylation
similar principles underlie information transfer in the cytoplasm and nucleus of cells. When the extracellular growth factor PDGF binds to its receptor outside of a cell, it dimerizes the
receptor. The result is to create a high effective
molarity of one receptor tail in the vicinity of the
other. Since the tails have tyrosine kinase activi-
ties, their proximal relationship facilitates trans-
phosphorylation. These phosphorylations take
place within flexible regions of the tails, and the
Recruitment of signaling proteins leads to localized enzymatic activity
Recruitment of signaling proteins leads to localized enzymatic activity
phosphate groups complete binding sites for intracellular signaling proteins that have substrates that reside within the inner leaflet of the
plasma membrane. The docking of the lipid kinase Bromodomain PI3K, for example, facilitates the phosphorylation
of its substrate, the membrane component phos-
phatidyl inositol-4.5-bisphosphate. A series of
subsequent events all proceed by this type of
induced proximity, allowing the signal to even-
Plus additional docking sites for signaling proteins
Plus additional docking sites for chromatin proteins
tually reach the nucleus. The new insight is that induced proximity is the key to information trans-
fer within chromatin, and that docking sites created when the signal reaches chromatin in the nucleus mediate network
behavior. For example, the cytoplasmic signal is received in the form of a histone acetyl transferase (HAT), which deposits
an acetyl group on a specific lysine side chain of a nucleosome in the vicinity of a target gene. This completes a binding
site for a signaling protein SWI/SNF (pronounced switch-sniff) that, after docking, remodels its now nearby nucleosome
substrate. This ATP-driven motor protein mechanically loosens the nucleosome so that the transcription apparatus can
access the promoter of a target gene. Small-molecule-based investigations of both networks helped illuminate their
fundamental operating principles.
Nevertheless, there is an inevitable feeling of missing out on the front-line action. The second issue is related, but even more personal. Exploring biology with the ad hoc use of organic chemistry draws the chemist into the seductive world of modern biology. Faced with questions about the relative role of organic chemistry and molecular biology or genetics on any given research undertaking, and wondering about what might happen with the next research undertaking, it is perhaps inevitable for chemists to increase their reliance on biological tools. But this is precisely what can lead to the personal crisis: "Will I be able to rely on my organic chemistry skill set on the next project?" Or even worse: "Am I becoming a biologist?" (Actually, "Am I becoming `just another' biologist!") In 1997, this angst was more than balanced by the excitement of what was emerging as a set of opportunities to transition from an ad hoc phase to a systematic one. The key was for organic chemistry to set itself on a course that would allow it
to tackle any problem in biology, from the dissection of a pathway to the understanding of complex networks, and eventually even to global biology or molecular physiology (for example, what is the basis of memory and cognition?). In the resulting plan that emerged, the early challenges are proving to be largely of a purely chemical nature, although a merger with information sciences seems inevitable. Organic chemistry as an initiator of discovery. For organic chemistry to play an initiating role in biological investigations, it will be important for organic chemists to be able to direct effectively synthetic chemistry efforts toward a set of probes--smallmolecule modulators--of two sorts. In the first case, a chemist might want to prepare small molecules having overall properties never seen before. In technical terms, we say that such molecules occupy a currently poorly populated region of multidimensional, chemical descriptor space. (Chemical descriptors are computable properties of small molecules; examples include vol-
ume, charge, number of bonds with low barrier to rotation, etcetera.) By accessing these small molecules using synthesis, this virgin swath of chemical space can be interrogated. In the second case, a chemist might want to prepare small molecules targeted to a region of chemical space optimal for modulating an area of biological interest. Here, it will be important to understand the relationship of chemical space to multidimensional, biological descriptor space ("biological space"). These goals were the basis, as a first step, of an attempt to formalize a planning algorithm for diversity-oriented synthesis (DOS), analogous to retrosynthetic analysis in target-oriented synthesis (TOS).26 DOS was able to draw upon technical developments in combinatorial synthesis, which is most often applied inTOS. In combinatorial chemistry the goal is, beginning with a small-molecule probe (or more often, a drug lead), to design a synthesis aimed to densely populate the region of chemical space occupied by the probe/lead; in collo-
C&EN / MARCH 3, 2003 55
PERSPECTIVE SMALL-MOLECULE DIFFERENTIATION A branching DOS pathway leads to efficient syntheses of complex and skeletally diverse small molecules.30
Beginning in lower middle, aldehydes are converted to unsaturated alcohols, and then to unsaturated, cyclic boronic esters. The latter species can be converted to a wide variety of products having skeletal diversity. The overall process is analogous to the differentiation of early lineage cells to middle and then late lineage cells. Differentiation in the DOS pathway, in this particular example, is dictated by the selection of reagents ("reagent-encoded"). An alternative process ("substrate-encoded") is discussed in the text.
quial terms, "to make analogs." In contrast, in DOS, the goals are either to populate chemical space broadly, or to target broad swaths of chemical space empirically found to overlap with the biology space that characterizes an area of biology (for example, cell-cycle checkpoints) or disease (for example, cancer or diabetes). To practice DOS in the future, several developments are required. Chemists must master their understanding of reaction transformations and hone their skills in this new type of strategic planning. The latter is particularly challenging and requires a type of strategic planning unfamiliar to chemists practicing TOS. Specifically, chemists must design branched reaction pathways that provide structurally complex and skeletally diverse small molecules in only three or four transformations. To achieve the goals of targeting these products to broad regions of chemical space, the computer increasingly will become the synthetic organic chemist's best friend. Although it has not yet been achieved, we can envision computations that will facilitate an organic chemist's selection of the reagents, Building Blocks and appendages, and prioritization of conceived DOS pathways that target a swath of chemical space of interest. Even more challenging, computational methods and databases for re-
lating the chemical and biological descriptor spaces must be developed. These considerations led in 1997 to the creation of the Harvard Institute of Chemistry & Cell Biology (ICCB), most recently sponsored by the National Cancer Institute's (NCI) Initiative for Chemical Genetics (ICG).27 An early (but interactive) version of ChemBank28, an NCIsponsored suite of informatic tools and federated databases, has just been launched on the Internet ( edu/chembank).The goal of ChemBank is to provide life scientists unfettered access to the above-described tools. For example, ChemBank should allow life scientists in a remote lab to tailor a DOS pathway emanating from a student's efforts in the student's lab to their own needs.This would require analysis tools at ChemBank that relate the selection of reagents and appendages to the swaths of chemical or biological spaces of interest to the remote lab. In this way, ChemBank would exploit the inherent plasticity of DOS pathways. A primary goal of ICCB-ICG is to foster ChemBank, in part by the development of systematic ways to explore biology with small molecules, that is, the development of chemical genetics. A related goal is to be able to apply chemical genetics widely, analogous to the way that biochemistry
and genetics can be applied to the dissection or interrogation of nearly any aspect of biology. Where we stand in terms of earning the name "chemical genetics" will be discussed in the final section. Developing DOS as an effective means to populate chemical space. The efficient synthesis of complex small molecules has been accomplished repeatedly in both TOS and DOS through the use of consecutive (or at least coupled) complexity-generating reactions.26,29 Likewise, it has been a conceptually (although not necessarily technically) simple matter to vary substituents on the resulting skeletons; the split-and-pool strategy using collections of appendages can provide an efficient solution. Far more challenging has been the conception of synthetic pathways leading to small molecules having a large variety of skeletons. Despite only limited success to date, several useful diversity-generating processes have emerged. DOS pathways are branched or divergent, in contrast to the generally linear or convergent pathways of TOS. One effective means of achieving skeletal diversity in these branched pathways uses "reagentencoded" processes. This strategy is reminiscent of the differentiation pathways seen in the expansion of pluripotent cells in biology (an example being a stem cell).
56 C&EN / MARCH 3, 2003
The early lineage cell (or stem cell) can differentiate into numerous middle lineage cells under the action of distinct differentiation factors.This process is repeated with the middle lineage cells at numerous stages, leading eventually into terminally differentiated cells (an example being a neuron). To emulate such highly branched biological pathways that lead to highly diverse products, organic chemists begin with their equivalent of a stem cell--a "pluripotent" organic functionality. Popular examples in my lab include aldehydes and terminal olefins. An example of a less useful functionality--at least given current capabilities in organic synthesis--closer to a "terminally differentiated state" is a methyl group lacking an adjacent activating group. Pluripotent functionality can be treated with many different reagents (analogous to the differentiation factors) to yield products having many different skeletons and, most importantly, that themselves are subject to the actions of different reagents yielding second, third, and subsequent layers of products having different skeletons. This differentiating process is illustrated on page 56, where the central aldehyde is first treated with nucleophilic reagents yielding unsaturated, secondary alcohols. This second core of functionality is treated with various unsaturated organoboronic ester reagents and a ruthenium catalyst resulting in a variety of skeletally distinct, cyclic and unsaturated organoboronic esters, which themselves are subject to the actions of a variety of electrophilic reagents, including oxidants and aldehydes.30 Appending processes can be added to the DOS pathway leading in a more straightforward way to diversity, although generally not to skeletal diversity. By analogy, a parallel line of thought leads to branched pathways having substrate-encoded elements; that is, the middle layers of products have structural information encoded within their structures that dictate the skeletons they are capable of obtaining, with many such substrates acquiring their unique final skeleton even under one single condition. Although such diversity-generating processes have not yet been demonstrated, they are under active investigation and appear to be a fertile (and intellectually challenging) area of research (unpublished results of work done by students in my laboratory, Martin D. Burke and Eric Berger). DOS and information science. Despite these conceptual and experimental advances, the field of DOS has not yet come close to reaching its goals. Although short and efficient DOS pathways have yielded complex and diverse small molecules with
novel biological properties22,31­37 (page At an even more primitive level, the scien-
59), the Structural Diversity is misleading. tific community has not yet agreed upon
Each small molecule depicted derives common standards and an ontology (con-
from its own DOS pathway. Even a quali- sistent and logical language) that will allow
tative analysis of the members emanating us to compare structures of small molecules
from a given pathway reveals that they are and their performance in biological assays.
disappointingly similar (a notion that has Despite the as-yet-unsolved problems in
been quantitated by computing molecu- this field, there are many reasons to be op-
lar descriptors for each compound and timistic about its future. The field has ma-
comparing them with compounds derived tured to a point where its shortcomings can
from the same versus different pathways). be identified, and there are encouraging
Of even greater concern is that the selec- signs of solutions looming on the horizon.
tion of compounds has so far been guided Funding agencies have responded in a dra-
only by the organic chemist's knowledge of matic fashion, and new and exciting re-
candidate reactions, creativity in planning search and training centers are now dotting
DOS pathways, and intuition about the the landscape.The merging of engineering
properties likely to yield effective modu- andinformationsciencewithorganicchem-
lators. Retrospectiveanalysesofthesecom- istry is already having a large effect. Infor-
pounds show that they tend to cluster in mation science is already being used to ad-
discrete regions of multidimensional de- dress the synthetic chemistry challenge
scriptor space.Although algorithms exist to noted above. ChemBank has as one of its
identify subsets of actual or virtual com- missions the adoption of common stan-
pounds that best distribute in chemical dards (for example, a common chemical
space in a defined way (for
registration system) and
example, a Gaussian dis- The goals are to an ontology that will al-
tribution, or an even distribution in a region of
target broad
low the management and sharing of small-molecule-
swaths of chemical space most like- ly to harbor small mole- culesabletopenetratethe chemical space
derived data. We hope that ChemBank will be a planning and discovery
blood-brain barrier), these are of little value to the
tool for chemists and biologists worldwide, the
planningofDOS.Inorder found to overlap only necessities being a
with the biology tofacilitatetheplanningof DOS pathways, organic
computer and access to the Internet. Finally, we
chemists require algo- space that
only have to turn to our
characterizes an rithms that identify the small subset of theoretical
students to gain a real glimpse of the future. The
area of biology. reactions, reagents, build- ing blocks, and appen-
fates of research fields are more in the hands of ea-
dages that will yield prod-
ger young scientists en-
ucts distributed in chemical space in a teringthetrainingphaseoftheircareerthan
defined way.
senior scientists who might have a tenden-
Further complicating matters is that we cy to avoid areas representing less familiar-
currently know little about the relationship ity and greater uncertainty. In this light, the
of chemical space and biological space (oth- challenges of DOS, although formidable,
er than rare exceptions such as the re- would appear to be reachable in the near
quirements for passing the blood-brain future. young students see uncertainty as
barrier). One hundred years of organic opportunity.38 My personal experience is
chemistry has not yet delivered a broadly that they also excel at applying their cre-
distributed collection of small molecules ative potential to this fertile area.
that fairly represents the expansive regions
Making chemical genetics accessi-
of chemical space, so we cannot possibly ble: the development of discovery plat-
know which regions correlate most effec- forms. At ICCB-ICG, we have been de-
tively with various biological outcomes. veloping an integrated set of techniques
Medicinal chemists are all too familiar with aimed at systematizing the application of
this problem as they attempt to identify small molecules, including DOS-derived
predictors of "bioavailability." The scien- smallmolecules,tobiology.Chemistrytech-
tific community has not yet developed a nology platforms are key, as the products
systematic means to assess how various of DOS should be prepared in a way that
small molecules perform in a set of assays ensures their effective integration into
reasonably well suited for measuring mul- screening experiments and in a way that
tidimensional biological descriptor space. ensures high purity and accuracy of struc-
58 C&EN / MARCH 3, 2003
ture assignment. One chemistry technology platform, based on the "one bead/one stock solution" strategy39a,b, has been developed at ICCB-ICG and is serving its users satisfactorily. It is being refined continuously and in ways that continue to allow its adoption in typically resource-limited academic settings. Other platforms are being developed elsewhere that might also be accessible and effective. ChemBank will benefit from these different platforms, so long as common standards and language are adopted. In a similar way, we have developed several effective techniques for small-molecule screening, including cytoblot assays4,40, screening-by-imaging using cells and organisms41a,b, and small-molecule42 and protein43 microarrays. These techniques have already yielded new insights into biology. As an early example, in collaboration with ICCB codirector Timothy J. Mitchison and members of his lab, we used a cytoblot screen and screeningby-imaging to discover monastrol--the first small-molecule inhibitor of mitosis that does not target tubulin.41a Monastrol
and, more recently, a more potent DOSderived compound, were shown in the Mitchison lab to inhibit Eg5, a kinesin motor protein.41a This discovery provided a powerful probe of the functions of this motor protein and a new medical lead. More than 50 labs have performed more than 100 chemical genetic screens at ICCBICG, leading to many small-molecule probes and insights into biology. Moving toward a chemical genomics. Most of these studies have resulted from small-molecule interrogation of specific problems in biology. In the future, small molecules will be used to probe global biology; this is an especially fertile area for organic chemistry ("chemical genomics," analogous to chemical genetics). To facilitate such studies at ICCB-ICG, we have introduced the concept of and are building a new laboratory ("ICCB-Kendall Square") for small-molecule annotation39b and profiling40, additional key elements of ChemBank. Here, the outcome of all experiments tends to be more important than the outcome of any individual experiment. By analyzing the outcome of the
complete matrix of a given collection of small molecules screened against a large collection of proteins and a large collection of phenotypic assays, chemists can envision many exciting outcomes. These include the development of methods to profile biological states with small molecules; to identify the protein target of a small-molecule modulator identified in a phenotypic screen; and to understand the relationship between chemical and biological descriptors and, ultimately, chemical space and biological space. ASSESSMENT OF WHERE WE ARE AND OF PROSPECTS FOR THE FUTURE. Much like the field of DOS, the field of chemical genetics is in an early formative stage. Encouraging experiments have been recorded, challenges have been identified, and solutions are being pursued. The successes, however, have not yet matched the distinguished history of the "ad hoc stage" of small-molecule explorations of life science. However, as in the field of DOS, there are many reasons to feel optimistic about the future.
DIVERSITY-ORIENTED SYNTHESIS Structures of representative small molecules synthesized using DOS principles and prepared as 5-mM stock solutions using the one bead/one stock solution technology platform: secramine31 (specific modulator of protein trafficking out of the Golgi apparatus; secramine is one member of a total of 2,800 small molecules prepared using DOS); calmoduphilin32 (Kd = 0.12 uM/calmodulin; one member of 29,400 total); haptamide33 (inhibitor of Hap3p-mediated transcription; one member of 4,320 total); uretupamine34 (binds to Ure2p and activates Nil1p-mediated transcription; one member of 32,000 total)22; tetracycle (one member of 2,500 total); tubacin35 (tubulin deacetylase inhibitor; one member of 7,200 deacetylase-biased dioxanes); macrolide36 (one member of 36 total); macrocyclic biaryl37 (affects cardiovascular system during zebrafish development; enantiomer has no activity; one member of 1,412 total).
Macrocyclic biaryl C&EN / MARCH 3, 2003 59
For chemical genetics to reach its full chemical genetic screens, the researchers
potential, it must be embraced as a gener- haveuncoveredsmall-moleculeagonistsand
al tool for exploring biology.As an approach antagonists of hedgehog signaling. Mecha-
to dissecting biology, like the genomic ap- nistic studies of these probes revealed that
proach, it is not yet viewed in the same light hedgehog proteins, which act through the
as biochemical or genetic approaches.Two integral membrane protein Patched, de-re-
key challenges go hand in hand: Chemical press the G-protein coupled receptor
genetic methods must be ac-
Smoothened, which is tar-
cessible, and the concepts For chemical geted by the probes. These
behind chemical genetics must permeate the thinking
genetics to
studies point to the exciting possibility of endogenous,
of life scientists. Efforts to reach its full
address the former have been described throughout
potential, it
small-molecule regulators of the Smoothened proteins. An entire issue of the Journal
this perspective. Is the con- must be embraced as cept of interrogating biology with small molecules sinking
of Biology (December 2002) was recently devoted to these advances. Continuing with
a general tool into the mind-set of life sci- entists on a broader scale
the theme of development and differentiation, Helene
than in the past? Recent ev- for exploring Gilgenkrantz at the Cochin
idence suggests so. The past several years
Institute, Paris, and coworkers used small-molecule di-
have seen a surge both in the
merizers in mice to ablate
number of reports of biological systems be- andregenerate,inadose-dependentfashion,
ing dissected with small molecules and in liver cells (hepatocytes).46 In addition, Pe-
the number of institutional commitments ter Schultz, at Scripps Research Institute,
to establishing the requisite infrastructure. and coworkers in two separate studies used
In response to numerous queries, we have chemical genetic screens to identify small
posted on the ICCB-ICG website a how- moleculesthatreversemyotubeformation47
to guide for building an academic screen- and that promote the differentiation of
ing facility (Caroline Shamu at Harvard mesenchymal progenitor cells into an os-
University, teoblast lineage.48
screening/faq_hts_facility.htm). Last year, Cell signaling. Using the principle of
the Howard Hughes Medical Institute an- chemical genetic synthetic lethal screen-
nounced its plans to develop a new research ing40, Philip Leder, Stanley J. Korsmeyer,
campus, Janelia Farm in Leesburg,Va., with and colleagues at Harvard Medical School
chemical genetics as one of several central screened matched cell lines differing only
elements of its unique, integrated, and col- in their levels of expression of the Neu
laborative structure.At ICCB-ICG alone, oncogene.49 A small molecule was identi-
we have provided small molecules and per- fied that interfered with cell function only
formed screens for a rapidly (and some- in cells overexpressing Neu. Mechanistic
what alarmingly) growing number of labo- studies revealed that the small molecule
ratories nationwide. Basic biological targeted the mitochondrial proton gradi-
insights gained from chemical genetics ent.This is an exciting experiment both in
studies from many labs worldwide signal a design and outcome. That an oncogene
noteworthy trend in the past several years. renders cells sensitive to disruptors of
To illustrate, I have selected several repre- mitochondrial function reveals a previous-
sentative examples from just two fields: de- ly hidden facet of cancer cell circuitry.That
velopmental biology and cell signaling.
mitochondria are integral components of
Developmental biology. Develop- the apoptotic signaling network is well ap-
mental biologists uncovered the hedge- preciated.However,insightsintotheirfunc-
hog-signaling network, including an outline tional role in signaling were gained in early
of its downstream signaling elements.This 2003 when a new apoptotic signaling on-
pathway is involved in the development of coprotein was discovered as the target of a
animals and the maintenance and repair of small-molecule modulator, which was bril-
adult cells, and its disruption results in cer- liantly uncovered using another chemical
tain types of cancer. The mysteries of the geneticscreen.50 Downstream of these pro-
pathway's mechanistic details are deepen- teins lies the key signal integrating protein
ing. Scientists at Curis Inc.44 and in Philip p53. Using protein-binding screens, scien-
A. Beachy's lab45 at Johns Hopkins dis- tists at Pfizer identified a small molecule
sected the pathway with an open embrace that stabilizes the folding of mutant forms
of genetic principles, yet using small mole- of DNA-binding p53; these probes are il-
cules as the source of perturbation. Using luminating the role of oncogenic muta-
tions in cancer and the possibility of reversing their consequences through biophysical means.51 To explore the function of another nucleic acid-protein interaction, Peter Beal and coworkers at the University of Utah identified a small-molecule probe of PKR, an RNA-dependent protein kinase thought to survey cells for evidence of viral invasion.52 Finally, in a study that links signaling to the previously discussed cytoskeleton, fascinating insights have been gained into a signaling pathway that regulates actin nucleation and polymerization.Acyclicpeptide53a and the small molecule wiskostatin53b were identified at Harvard Medical School by Marc W. Kirschner and colleagues in chemical genetic screens and found to bind to the neuronal-Wiskott Aldrich Syndrome Protein (N-WASP). Wiskostatin binding to NWASP stabilizes an autoinhibited state and prevents N-WASP from activating a downstream actin-nucleating complex (Arp2/3), which functions to nucleate the actin polymer. The small-molecule probes revealed a previously unidentified mechanism for targeting interactions between key proteins in cells.53 IN SUMMARY, the systematic population of chemical space with small molecules using DOS and information science, the systematic probing of biology space with these small molecules, and the sharing of results and data using common standards and language on public databases promise a future understanding of the relationship of chemical and biological spaces. This is an exciting and important prospect, and one that will require organic chemistry to play a discovery role. Synthetic organic chemists in particular will need to meet the challenges of DOS. With such an understanding and with the availability of appropriate tools, organic chemists may in the future design synthetic pathways yielding small molecules targeted to the swath of chemical space optimal for modulating the swath of biological space of interest. Although different in its reliance on empirical observations that permit "targeting," the desired capabilities are reminiscent of those provided by natural selection, the process that yields natural products. A noteworthy difference is the anticipated time frame of the former relative to the latter! Stuart L. Schreiber is an investigator with the Howard Hughes Medical Institute and is chair of the Department of Chemistry and chemical biology at Harvard University. He is a founder of the Harvard Institute of Chemistry & Cell Biology­Initiative for Chemical Genetics.
60 C&EN / MARCH 3, 2003
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29. For example, see: Lee, D. et al. Org. Lett. 2 (2000): 709­712. 30. (a) Micalizio, G. C., and S. L. Schreiber. Angew. Chem. Int. Ed. 41 (2002): 152­154. (b) Micalizio, G. C., and S. L. Schreiber. Angew. Chem. Int. Ed. 41 (2002): 3272­3276. 31. Pelish, H. E. et al. J. Am. Chem. Soc. 123 (2001): 6740­6741. 32. Kwon, O. et al. J. Am. Chem. Soc. 124 (2002): 13402­13404. 33. (a) Stavenger, R. A., and S. L. Schreiber. Angew. Chem. Int. Ed. 40 (2001): 3417­3421. (b) Koehler, A., and S. L. Schreiber. Manuscript in preparation. 34. Kubota, H. et al. Chem. Biol. 9 (2002): 265­276. 35. (a) Sternson, S. M. et al. Org. Lett. 3 (2001): 4239­4242. (b) Haggarty, S. J. et al. Proc. Natl. Acad. Sci. USA, in press. 36. Schmidt, D. et al. J. Comb. Chem., in press. 37. (a) Spring, D. R. et al. J. Am. Chem. Soc. 122 (2000): 5656­5657. (b) Spring, D. R. et al. J. Am. Chem. Soc. 124 (2002): 1354­1363. 38. Burke, M. D., and G. Lalic. Chem. Biol. 9 (2002): 535­541. 39. (a) Blackwell, H. E. et al. Chem. Biol. 8 (2001): 1167­1182. (b) Clemons, P. A. et al. Chem. Biol. 8 (2001): 1183­1195. (c) Blackwell, H. E. et al. Angew. Chem. Int. Ed. 40 (2001): 3421­3425. 40. Stockwell, B. R. et al. Chem. Biol. 6 (1999): 71­83. 41. (a) Mayer, T. U. et al. Science 286 (1999): 971­974. (b) Peterson, R. T. et al. Proc Natl. Acad. Sci. USA 97 (2000): 12965­12969. 42. (a) MacBeath, G. et al. J. Am. Chem. Soc. 121 (1999): 7967­7968. (b) Hergenrother, P. J. et al. J. Am. Chem. Soc. 122 (2000): 7849­7850. (c) Barnes-Seeman, D. Angew. Chem. Int. Ed., in press. 43. MacBeath, G., and S. L. Schreiber. Science 289 (2000): 1760­1762. 44. Frank-Kamenetsky, M. et al. J. Biol. 1 (2002): 1:10. 45. Chen, J. K. et al. Proc. Natl. Acad. Sci. USA 99 (2002): 14071­14076. 46. Mallet, V. O. et al. Nat. Biotechnol. 20 (2002): 1234­1239. 47. (a) Rosania, G. R. et al. Nat. Biotechnol. 18 (2000): 304. (b) For commentary, see: Frederickson, R. Nat. Biotechnol. 18 (2000): 250. 48. Wu, X. et al. J. Am. Chem. Soc. 124 (2002): 14520­14521. 49. Fantin, V. R. et al. Cancer Cell 2 (2002): 29­42. 50. (a) Jiang, X. et al. Science 299 (2003): 223­226. (b) Nicholson, D. W., and N. A. Thornberry. Science 299 (2003): 214­215. 51. Foster, B. A. et al. Science 286 (1999): 2507­2510. 52. Carlson, C. B. et al. ChemBioChem 3 (2002): 859­865. 53. (a) Peterson, J. R. et al. Proc. Natl. Acad. Sci. USA 98 (2001): 10624­10629. (b) Peterson, J. R. et al. Unpublished results and ref. 3(b).
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