THE SMALL-MOLECULE APPROACH to BIOLOGY Chemical Genetics and Diversity-Oriented Organic Synthesis Make Possible the Systematic Exploration of Biology

PERSPECTIVE IMAGE BY CATHY NEWTON AND ERIC KELLER, HOWARD HUGHES MEDICAL INSTITUTE

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 ing similarities between the small-molecule and genetic approaches have increased mall molecules have long been associated the generality of the small-molecule approach. Although a truly systematic way with biological discoveries, but, in contrast to bio- to explore any and all facets of biology with chemical and genetic approaches, the small-mole- small-molecule modulators has not yet been reached, these advances are begin- cule approach has lacked generality. Although the ning to influence researchers on a much advances made through the use of small molecules broader scale. It is becoming more common for a life scientist to ask, “Should I Sas probes (as distinct from medicines) are impressive, they have tackle this problem with small molecules?” or even to state, “The only way I can tack- in general come about on a case-by-case basis. Advances in di- le this problem is with small molecules.”1 versity-oriented organic synthesis and a focus on the underly- The latter is becoming increasingly com-

HTTP://WWW.CEN– ONLINE.ORG C&EN / MARCH 3, 2003 51 PERSPECTIVE mon as global views of biology are sought. of gene mutations, either naturally occur- Cytoskeletal research in general has been The discovery principles and platforms ring, randomly induced, or targeted. They a rich beneficiary of small-molecule enabling this transformation constitute have developed powerful analysis tools, probes.3b Microtubule-stabilizing agents what Rebecca Ward at Harvard Universi- such as “epistasis analysis” to order genes in such as paclitaxel (Taxol) played an impor- ty first coined the “chemical genetic” ap- pathways, “synthetic lethal screening” to tant role in the identification of micro- proach on the cover of the inaugural issue reveal redundant elements of pathways and tubule-associated proteins (MAPs). Actin- of Chemistry & Biology nine years ago. Her networks, and “modifier (suppressor and disrupting agents such as cytochalasin and term reminds us that, to understand a life enhancer) screening” to reveal connections latrunculin have played key roles in unrav- process, you should perturb it and deter- between pathways and networks. These eling the mysteries of the actin cytoskeleton, mine the consequence and that such an ap- principles and analysis tools are directly ap- and recently discovered small molecules proach should strive to have the broad gen- plicable to chemical genetics. Here, of that specifically target motor proteins that erality and power of genetics. That is, it course, the wild-type protein is used; it is the carry their cargo along the actin and mi- should allow the probing of life processes binding of a small molecule to the protein crotubule polymers are now being used to in both a systematic and thorough way (anal- that results in a perturbation of function, reveal previously hidden facets of motor ogous to the application of genetics and to either inhibition or activation. Recognizing function.3b Recent experiments concern- the use of saturation mutagenesis, respec- this parallel alone, however, does not en- ing the regulation of the cytoskeleton sug- tively). Chemical genetics is a logical out- sure a general approach to exploring biol- gest that the use of small molecules to un- growth and subset of chemical biology, ogy. Aresearch infrastructure involving new derstand this area of research will continue where chemical principles and techniques advances in chemistry must be developed in the future. are used to dissect directly, rather than to and integrated into the fabric of day-to-day Ion channels and signaling in the model, biology. In this perspective, I aim to life science research. It must be routine and neurosciences. Neurobiology has long focus on the current transition from the ad readily available to life science researchers, been a beneficiary of the ability of small hoc to systematic use of small molecules to especially to chemists and biologists. In the molecules to target the neurotransmitter explore the life sciences (rather than to dis- sections of this perspective, I describe the receptors and ion channels that function cover new medicines) and the role of or- use of small molecules that have illuminat- in neurons. As a result, neurobiologists tend ganic chemistry in mediating this transition. ed life processes, the shortcomings of this to be among the most eager to see advances The same experiments that encouraged type of research as a general approach, ef- in chemistry relevant to small molecules. me to explore biology with organic chem- forts to overcome these shortcomings, and Natural products, especially from snake, istry frustrated me as well. These frustra- an assessment of where we stand today. spider, bee, scorpion, dinoflagellate, pep- tions had to do with, at the time, the in- per, snail, puffer fish, and soft coral, have ability of the small-molecule approach (in AD HOC USE OF SMALL MOLECULES TO played a particularly prominent role in these this context, sometimes referred to as the EXPLORE BIOLOGY.The past century has studies.5 Scientists at Pfizer, recognizing “pharmacological approach”) to be applied yielded many examples of researchers iden- the treasure trove of ion channel probes with the broad generality of the reduc- tifying and using small molecules to probe stemming historically from spiders, recently tionist-based biochemical and discovery- aspects of biology. Some of these served as developed a fruitful effort in both the iso- based genetic approaches. As a result, I was the subject of this article’s prequel, au- lation of channel-blocking natural products left with an uneasy feeling about the role of thored 10 years earlier.2 from spiders and the synthesis of optimized organic chemistry in future studies. Asecure Cytoskeleton. Gary G. Borisy and Edwin variants. These small molecules were used role for organic chemistry would entail its W. Taylor’s use of colchicine, at the MRC to classify channel subtypes and to probe use “front and center” as a general discov- Laboratory of Molecular Biology, in Cam- their functions in neurobiology. ery engine, rather than relying on chance bridge, England, to identify the tubulin pro- At the National Institutes of Health, opportunities provided by neighboring dis- teins is a classic illustration of using small Arvid Carlsson’s use of reserpine, L-DOPA, ciplines. Overcoming these frustrations re- molecules for discovery in basic biology.3 Col- and chlorpromazine led to the discovery of quired adapting the principles that under- chicine, along with numerous recently dis- the neurotransmitter dopamine and to its lie genetics (and more recently genomics) covered small molecules, targets the -tub- role in mediating signals within the ner- to chemistry—a process that is proving to ulin/-tubulin protein-protein interface of vous system (“chemical transmission in the be a fertile one for chemistry. For example, microtubules and thus disrupts microtubules brain”).6 His studies of dopamine and the much as natural products have driven the in cells. Despite the widely held view that dopamine receptor, and of antagonists of development of both target-oriented syn- small molecules generally fail to disrupt pro- their interaction such as chlorpromazine, thesis (TOS) and synthetic methods, chem- tein-protein interactions, this interaction ap- provided early hints of how extracellular ical genetics is providing a driving force for pears to be a particularly simple one. For ex- factors, without entering a cell, can give the development of diversity-oriented syn- ample, in one small-molecule screen, over rise to changes in intracellular processes— thesis (DOS; see below). 300 of 16,000 small molecules were shown in other words, signal transduction. For From a century of genetics-based inter- to have this property, while two were found these discoveries, Carlsson was awarded a rogations of life, we have learned that per- to be stabilizers of the same protein-protein share of the Nobel Prize in Physiology or turbing life processes and observing the interaction.4 Such molecules have illumi- Medicine in 2000. consequences can provide illuminating in- nated the functions of microtubules as key Inner leaflet of the plasma mem- sights. Geneticists do so through the use cytoskeletal elements. 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 HTTP://WWW.CEN– ONLINE.ORG Colchicine Spidamine Reserpine Phorbol

Somatostatin Pioglitazone MK-886 receptor agonist 506BD

Dimerizer (bumped rapamycin) 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 es using small-molecule probes. As with all abetes, although that role is still mysteri- key functions of members of a large fami- studies using small molecules, two ap- ous, as is the molecular etiology of the dis- ly of protein kinases named “PKCs” in in- proaches have been used: One emulates ease. A second example of this approach tracellular signal transduction.7 They also the underlying principles of classical ge- derived from research aimed at under- revealed a docking site used by these pro- netics (sometimes called “forward genet- standing the molecular basis of inflamma- teins to associate with the inner leaflet of ics”) and the other a modern variant of it, tion. In mechanistic studies of the anti-in- the plasma membrane. Phorbol diesters reverse genetics. flammatory agent MK-886, this small bind to the PKCs and tether them to the As an illustration of forward chemical molecule was used to discover 5-lipoxyge- leaflet, thereby creating proximal rela- genetics, small molecules were screened at nase-activating protein (FLAP) and to as- tionships with their leaflet-localized sub- many companies in the 1970s in search of sign its cellular and physiological func- strates. These studies demonstrate how agents capable of treating type 2 diabetes. tions.9 These studies opened the door to small molecules can activate the functions Pioglitazone, the archetype of the “glita- a new area of research involving FLAPand of the proteins to which they bind by di- zones,” was discovered at Takeda Chemi- its role in inflammation. recting them to specific locales within cells. cal Industries, in Japan, during this period. As an illustration of reverse chemical Biological insights stemming from Only in more recent years has the target of genetics, small molecules have also been pharmaceutical research. Researchers the glitazones been determined. In accor- identified that selectively bind and acti- in the pharmaceutical and biotechnology dance with insights gained from human vate five members (paralogs) of the so- industries, while in search of new medi- genetics, the glitazones bind and activate matostatin receptor family. Having prede- cines, have been particularly effective at the nuclear receptor PPAR.8 It is now termined the selectivity of these probes discovering new insights into life process- known that PPAR plays a key role in di- toward individual paralogs, scientists at

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Merck were able to uncover the distinctive through proximity effects. We demon- By taking a global approach to under- functions of the paralogs.10 This remark- strated that small molecules could be used standing chromatin function, we recently able investigation used the logic of reverse to influence signaling pathways in an ani- proposed a “signaling network model” of genetics, in that the researchers targeted mal with temporal and spatial control.18 chromatin and compared it with an alter- the modulation of function (in this case, Subsequently, many researchers working native view, the “histone code hypothesis” the more challenging activation of func- on many research problems have had suc- presented by Allis.25 tion) of individual paralogs, then searched cess with this approach, and dimerizer kits Research by many scientists in this area broadly for the resulting consequences. have now been distributed freely to more has shined a bright light on chromatin as Signaling networks in the cytoplasm than 500 laboratories by Ariad Pharma- a key regulatory element, rather than sim- and nucleus. In my laboratory in the ear- ceuticals.19 Its promise in gene therapy has ply a structural element, in transcription. ly and mid-1980s, a focus on target-ori- been highlighted by the stable (over sever- This research followed previous small-mol- ented synthesis and on developing syn- al years), small-molecule-induced produc- ecule-based studies of signal transduction thetic methods eventually led to studies tion of erythropoeitin (EPO) in primates that helped reveal the existence of cyto- of how the small-molecule objects of our by treating primates with a small-molecule plasmic signaling networks. The signaling studies perturb the functions of the pro- switch for an EPO-inducing transcription network model of chromatin25 posits that teins to which they bind. These studies us- factor, and more recently, in Phase II hu- both cytoplasmic signaling and chromatin ing natural products allowed us to discov- man clinical trials for treatment of graft- signaling use key elements of networks, in- er new principles of biological signaling versus-host disease. cluding feedback motifs and redundancy networks, including the commonality of Members of my group and of Solomon that ensure robustness, adaptability, and principles underlying both cytoplasmic and H. Snyder’s group at Johns Hopkins Uni- switchlike behavior. This insight was gained nuclear signaling networks. Four studies versity independently discovered in 1994 in part by recognizing the remarkable sim- of natural products revealed basic insights that the small molecule rapamycin simul- ilarities in the principles that underlie in- into information transfer in biology. taneously binds FKBP12 and the previ- formation transfer in the cytoplasm and The discovery of the FK506-binding ously unknown protein we named FRAP nucleus (page 55). Our view of information protein FKBP12 in 1988 (independently (FKBP12-rapamycin binding protein, also transfer in cells has been extended from discovered in my lab and by scientists at known as TORand RAFT).20 Using chem- the early events of signal detection, often Merck)11 was facilitated by target-oriented ical genetic epistasis analysis21, diversity- at the plasma membrane, to chromatin, synthesis efforts aimed at, among others, oriented synthesis, and small-molecule mi- where memory of the signal is established the natural products FK506 and rapamy- croarrays, among other techniques, we in nondividing cells, and inheritance of the cin and the nonnatural small molecules succeeded in uncovering the nutrient-re- signal (epigenetics) is achieved in dividing 506BD and tricyclosporin.12 Using a num- sponse signaling network involving TOR cells. The concept of signaling pathways ber of these small-molecule probes, we proteins in yeast and FRAP/TORin mam- (in my opinion, an artifact of the reduction determined in 1991 that FK506 and cy- malian cells. Small molecules such as ure- approach) has been evolving toward the closporin inhibit the activity of the phos- tupamine22 and rapamycin were shown to concept of signaling networks. The fine phatase calcineurin. This occurs by an un- be particularly effective in illuminating the temporal control of protein function in usual mechanism: through the formation ability of proteins such as FRAP, Tor1p, cells afforded by small molecules has played of the ternary complexes FKBP12- Tor2p, and Ure2p to receive multiple inputs a key role in this transition.25 FK506-calcineurin and cyclophilin-cy- and to process them appropriately toward closporin-calcineurin.13 This work, to- multiple outputs (“multichannel proces- SYSTEMATIC USE OF SMALL MOLE- gether with work by Gerald R. Crabtree at sors”). The nutrient signaling network now CULES TO EXPLORE BIOLOGY: CHEMI- Stanford University concerning the NFAT appears to be key to understanding the ori- CAL GENETICS. From the perspective of proteins, led to the elucidation of the cal- gins of type 2 diabetes. an organic chemist participating in the cium-calcineurin-NFAT signaling path- In 1996, my lab used a synthetic vari- study of small-molecule-based signaling way.14 This proved to be an early example ant (an immobilized version of K-trap, page networks in 1997, I could not help but be of defining an entire cellular signaling path- 53) of the natural product trapoxin to mol- concerned with two issues. First, the skill way from the cell surface to the nucleus, ecularly characterize the histone deacetyl- set of an organic chemist was well suited analogous to that of the Ras-Raf-MAPK ases (HDACs).23 Prior to our work in this for responding to discovery opportunities pathway elucidated the following year. In area, the HDAC proteins had not been iso- provided by biologists, but not necessari- subsequent years, the central roles of the lated—despite many attempts by others ly for initiating or leading the discovery calcium-calcineurin-NFATsignaling path- who were inspired by Vincent G. Allfrey’s program. This is, in retrospect, not sur- way in immune function, heart develop- detection, at Rockefeller University, of the prising, as it mirrors the role of organic ment, and memory acquisition were re- enzymatic activity in cell extracts more chemists in the process of modern drug vealed by many researchers working in a than 30 years earlier. Coincident with the discovery. In contrast to drug discovery variety of fields.15 HDAC discovery, C. David Allis and col- prior to the mid-1970s, with the advent of The ability of small molecules to bind leagues at the University of Rochester, tak- molecular biology and molecular cell biol- two proteins simultaneously inspired our ing a biochemical approach, reported their ogy, organic chemists are typically asked collaboration with Crabtree and members discovery of the histone acetyl transferas- to participate in the optimization process of his laboratory in 1993 to develop “small- es (HATs).24 These two contributions cat- following the decision by biologists to se- molecule dimerizers,” which were shown to alyzed much research in this area, eventu- lect a specific biological target for thera- provide small-molecule regulation of tran- ally leading to the characterization of peutic intervention. This concern is ad- scription and of numerous signaling mole- numerous histone-modifying enzymes, mittedly a selfish one—there should be no cules and pathways (for example, the Fas, their resulting histone “marks,” and nu- problem with biology driving science un- insulin, TGF, and T-cell receptors16, 17 ) merous proteins that bind to these marks. less perhaps you happen to be a chemist!

54 C&EN / MARCH 3, 2003 HTTP://WWW.CEN– ONLINE.ORG SIGNALING NETWORKS Small-molecule- based investigations of membrane-to-nucleus signaling and chromatin function suggest the existence of network motifs that ensure robustness, adaptability, and switchlike behavior, as Kinase well as other striking commonalities (the signal- subdomains ing network model of chromatin).25 The figure uses the platelet-derived growth factor receptor Tail (PDGFR) and a nucleosome to illustrate that Kinase Tail similar principles underlie information transfer in insert the cytoplasm and nucleus of cells. When the Extracellular signal leads Nuclear signal leads to receptor dimerization, to lysine acetylation extracellular growth factor PDGF binds to its trans-phosphorylation receptor outside of a cell, it dimerizes the receptor. The result is to create a high effective

IMAGE COURTESY OF STUART SCHREIBER, HARVARD UNIVERSITY 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 Recruitment of signaling phosphate groups complete binding sites for proteins leads to localized proteins leads to localized intracellular signaling proteins that have sub- enzymatic activity enzymatic activity strates 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 Plus additional docking sites tually reach the nucleus. The new insight is that for signaling proteins for chromatin proteins induced proximity is the key to information transfer 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 to tackle any problem in biology, from the ume, charge, number of bonds with low of missing out on the front-line action. dissection of a pathway to the under- barrier to rotation, etcetera.) By accessing The second issue is related, but even standing of complex networks, and even- these small molecules using synthesis, this more personal. Exploring biology with the tually even to global biology or molecular virgin swath of chemical space can be in- ad hoc use of organic chemistry draws the physiology (for example, what is the basis terrogated. In the second case, a chemist chemist into the seductive world of mod- of memory and cognition?). In the result- might want to prepare small molecules tar- ern biology. Faced with questions about the ing plan that emerged, the early challenges geted to a region of chemical space opti- relative role of organic chemistry and mo- are proving to be largely of a purely chem- mal for modulating an area of biological lecular biology or genetics on any given re- ical nature, although a merger with infor- interest. Here, it will be important to un- search undertaking, and wondering about mation sciences seems inevitable. derstand the relationship of chemical space what might happen with the next research Organic chemistry as an initiator of to multidimensional, biological descriptor undertaking, it is perhaps inevitable for discovery. For organic chemistry to play space (“biological space”). chemists to increase their reliance on bio- an initiating role in biological investigations, These goals were the basis, as a first step, logical tools. But this is precisely what can it will be important for organic chemists to of an attempt to formalize a planning algo- lead to the personal crisis: “Will I be able be able to direct effectively synthetic chem- rithm for diversity-oriented synthesis to rely on my organic chemistry skill set on istry efforts toward a set of probes—small- (DOS), analogous to retrosynthetic analy- the next project?” Or even worse: “Am I be- molecule modulators—of two sorts. In the sis in target-oriented synthesis (TOS).26 coming a biologist?” (Actually, “Am I be- first case, a chemist might want to prepare DOS was able to draw upon technical de- coming ‘just another’ biologist!”) small molecules having overall properties velopments in combinatorial synthesis, In 1997, this angst was more than bal- never seen before. In technical terms, we say which is most often applied in TOS. In com- anced by the excitement of what was that such molecules occupy a currently binatorial chemistry the goal is, beginning emerging as a set of opportunities to tran- poorly populated region of multidimen- with a small-molecule probe (or more often, sition from an ad hoc phase to a systemat- sional, chemical descriptor space. (Chem- a drug lead), to design a synthesis aimed to ic one. The key was for organic chemistry ical descriptors are computable properties densely populate the region of chemical to set itself on a course that would allow it of small molecules; examples include vol- space occupied by the probe/lead; in collo-

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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, lating the chemical and biological descrip- and genetics can be applied to the dissec- in DOS, the goals are either to populate tor spaces must be developed. tion or interrogation of nearly any aspect chemical space broadly, or to target broad These considerations led in 1997 to the of biology. Where we stand in terms of swaths of chemical space empirically found creation of the Harvard Institute of Chem- earning the name “chemical genetics” will to overlap with the biology space that char- istry & Cell Biology (ICCB), most recent- be discussed in the final section. acterizes an area of biology (for example, ly sponsored by the National Cancer In- Developing DOS as an effective cell-cycle checkpoints) or disease (for ex- stitute’s (NCI) Initiative for Chemical means to populate chemical space.The ample, cancer or diabetes). Genetics (ICG).27 An early (but interac- efficient synthesis of complex small mol- To practice DOS in the future, several tive) version of ChemBank28, an NCI- ecules has been accomplished repeatedly developments are required. Chemists must sponsored suite of informatic tools and in both TOS and DOS through the use of master their understanding of reaction federated databases, has just been launched consecutive (or at least coupled) complex- transformations and hone their skills in this on the Internet (http://iccb.med.harvard. ity-generating reactions.26,29 Likewise, it new type of strategic planning. The latter edu/chembank). The goal of ChemBank is has been a conceptually (although not nec- is particularly challenging and requires a to provide life scientists unfettered access essarily technically) simple matter to vary type of strategic planning unfamiliar to to the above-described tools. For example, substituents on the resulting skeletons; the chemists practicing TOS. Specifically, ChemBank should allow life scientists in a split-and-pool strategy using collections of chemists must design branched reaction remote lab to tailor a DOS pathway ema- appendages can provide an efficient solu- pathways that provide structurally com- nating from a student’s efforts in the stu- tion. Far more challenging has been the plex and skeletally diverse small molecules dent’s lab to their own needs. This would re- conception of synthetic pathways leading in only three or four transformations. To quire analysis tools at ChemBank that relate to small molecules having a large variety of achieve the goals of targeting these prod- the selection of reagents and appendages to skeletons. Despite only limited success to ucts to broad regions of chemical space, the the swaths of chemical or biological spaces date, several useful diversity-generating computer increasingly will become the syn- of interest to the remote lab. In this way, processes have emerged. thetic organic chemist’s best friend. Al- ChemBank would exploit the inherent plas- DOS pathways are branched or diver- though it has not yet been achieved, we can ticity of DOS pathways. gent, in contrast to the generally linear or envision computations that will facilitate Aprimary goal of ICCB-ICG is to fos- convergent pathways of TOS. One effec- an organic chemist’s selection of the ter ChemBank, in part by the development tive means of achieving skeletal diversity in reagents, building blocks and appendages, of systematic ways to explore biology with these branched pathways uses “reagent- and prioritization of conceived DOS path- small molecules, that is, the development encoded” processes. This strategy is rem- ways that target a swath of chemical space of chemical genetics. A related goal is to iniscent of the differentiation pathways of interest. Even more challenging, com- be able to apply chemical genetics widely, seen in the expansion of pluripotent cells putational methods and databases for re- analogous to the way that biochemistry in biology (an example being a stem cell).

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The early lineage cell (or stem cell) can dif- novel biological properties22,31–37 (page At an even more primitive level, the scien- ferentiate into numerous middle lineage 59), the structural diversity is misleading. tific community has not yet agreed upon cells under the action of distinct differen- Each small molecule depicted derives common standards and an ontology (con- tiation factors. This process is repeated with from its own DOS pathway. Even a quali- sistent and logical language) that will allow the middle lineage cells at numerous stages, tative analysis of the members emanating us to compare structures of small molecules leading eventually into terminally differ- from a given pathway reveals that they are and their performance in biological assays. entiated cells (an example being a neuron). disappointingly similar (a notion that has Despite the as-yet-unsolved problems in To emulate such highly branched biologi- been quantitated by computing molecu- this field, there are many reasons to be op- cal pathways that lead to highly diverse lar descriptors for each compound and timistic about its future. The field has ma- products, organic chemists begin with their comparing them with compounds derived tured to a point where its shortcomings can equivalent of a stem cell—a “pluripotent” from the same versus different pathways). be identified, and there are encouraging organic functionality. Popular examples in Of even greater concern is that the selec- signs of solutions looming on the horizon. my lab include aldehydes and terminal tion of compounds has so far been guided Funding agencies have responded in a dra- olefins. An example of a less useful func- only by the organic chemist’s knowledge of matic fashion, and new and exciting re- tionality—at least given current capabili- candidate reactions, creativity in planning search and training centers are now dotting ties in organic synthesis—closer to a “ter- DOS pathways, and intuition about the the landscape. The merging of engineering minally differentiated state” is a methyl properties likely to yield effective modu- and information science with organic chem- group lacking an adjacent activating group. lators. Retrospective analyses of these com- istry is already having a large effect. Infor- Pluripotent functionality can be treated pounds show that they tend to cluster in mation science is already being used to ad- with many different reagents (analogous to discrete regions of multidimensional de- dress the synthetic chemistry challenge the differentiation factors) to yield prod- scriptor space. Although algorithms exist to noted above. ChemBank has as one of its ucts having many different skeletons and, identify subsets of actual or virtual com- missions the adoption of common stan- most importantly, that themselves are sub- pounds that best distribute in chemical dards (for example, a common chemical ject to the actions of different reagents space in a defined way (for registration system) and yielding second, third, and subsequent lay- example, a Gaussian dis- The goals are to an ontology that will al- ers of products having different skeletons. tribution, or an even dis- low the management and This differentiating process is illustrated tribution in a region of target broad sharing of small-molecule- on page 56, where the central aldehyde is chemical space most like- derived data. We hope first treated with nucleophilic reagents ly to harbor small mole- swaths of that ChemBank will be a yielding unsaturated, secondary alcohols. cules able to penetrate the chemical space planning and discovery This second core of functionality is treat- blood-brain barrier), these tool for chemists and bi- ed with various unsaturated organoboronic are of little value to the empirically ologists worldwide, the ester reagents and a ruthenium catalyst re- planning of DOS. In order found to overlap only necessities being a sulting in a variety of skeletally distinct, to facilitate the planning of computer and access to cyclic and unsaturated organoboronic es- DOS pathways, organic with the biology the Internet. Finally, we ters, which themselves are subject to the chemists require algo- space that only have to turn to our actions of a variety of electrophilic reagents, rithms that identify the students to gain a real including oxidants and aldehydes.30 Ap- small subset of theoretical characterizes an glimpse of the future. The pending processes can be added to the DOS reactions, reagents, build- fates of research fields are pathway leading in a more straightforward ing blocks, and appen- area of biology. more in the hands of ea- way to diversity, although generally not to dages that will yield prod- ger young scientists en- skeletal diversity. By analogy, a parallel line ucts distributed in chemical space in a tering the training phase of their career than of thought leads to branched pathways hav- defined way. senior scientists who might have a tenden- ing substrate-encoded elements; that is, the Further complicating matters is that we cy to avoid areas representing less familiar- middle layers of products have structural currently know little about the relationship ity and greater uncertainty. In this light, the information encoded within their struc- of chemical space and biological space (oth- challenges of DOS, although formidable, tures that dictate the skeletons they are ca- er than rare exceptions such as the re- would appear to be reachable in the near pable of obtaining, with many such sub- quirements for passing the blood-brain future. Young students see uncertainty as strates acquiring their unique final skeleton barrier). One hundred years of organic opportunity.38 My personal experience is even under one single condition. Although chemistry has not yet delivered a broadly that they also excel at applying their cre- such diversity-generating processes have distributed collection of small molecules ative potential to this fertile area. not yet been demonstrated, they are under that fairly represents the expansive regions Making chemical genetics accessi- active investigation and appear to be a fer- of chemical space, so we cannot possibly ble: the development of discovery plat- tile (and intellectually challenging) area of know which regions correlate most effec- forms. At ICCB-ICG, we have been de- research (unpublished results of work done tively with various biological outcomes. veloping an integrated set of techniques by students in my laboratory, Martin D. Medicinal chemists are all too familiar with aimed at systematizing the application of Burke and Eric Berger). this problem as they attempt to identify small molecules, including DOS-derived DOS and information science. Despite predictors of “bioavailability.” The scien- small molecules, to biology. Chemistry tech- these conceptual and experimental ad- tific community has not yet developed a nology platforms are key, as the products vances, the field of DOS has not yet come systematic means to assess how various of DOS should be prepared in a way that close to reaching its goals. Although short small molecules perform in a set of assays ensures their effective integration into and efficient DOS pathways have yielded reasonably well suited for measuring mul- screening experiments and in a way that complex and diverse small molecules with tidimensional biological descriptor space. ensures high purity and accuracy of struc-

58 C&EN / MARCH 3, 2003 HTTP://WWW.CEN– ONLINE.ORG ture assignment. One chemistry technolo- and, more recently, a more potent DOS- complete matrix of a given collection of gy platform, based on the “one bead/one derived compound, were shown in the small molecules screened against a large stock solution” strategy39a,b, has been de- Mitchison lab to inhibit Eg5, a kinesin mo- collection of proteins and a large collec- veloped at ICCB-ICG and is serving its tor protein.41a This discovery provided a tion of phenotypic assays, chemists can users satisfactorily. It is being refined con- powerful probe of the functions of this mo- envision many exciting outcomes. These tinuously and in ways that continue to allow tor protein and a new medical lead. More include the development of methods to its adoption in typically resource-limited than 50 labs have performed more than profile biological states with small mole- academic settings. Other platforms are be- 100 chemical genetic screens at ICCB- cules; to identify the protein target of a ing developed elsewhere that might also be ICG, leading to many small-molecule small-molecule modulator identified in a accessible and effective. ChemBank will probes and insights into biology. phenotypic screen; and to understand the benefit from these different platforms, so Moving toward a chemical genomics. relationship between chemical and bio- long as common standards and language are Most of these studies have resulted from logical descriptors and, ultimately, chemi- adopted. small-molecule interrogation of specific cal space and biological space. In a similar way, we have developed sev- problems in biology. In the future, small eral effective techniques for small-mole- molecules will be used to probe global bi- ASSESSMENT OF WHERE WE ARE AND cule screening, including cytoblot as- ology; this is an especially fertile area for or- OF PROSPECTS FOR THE FUTURE. says4,40, screening-by-imaging using cells ganic chemistry (“chemical genomics,” Much like the field of DOS, the field of and organisms41a,b, and small-molecule42 analogous to chemical genetics). To facili- chemical genetics is in an early formative and protein43 microarrays. These tech- tate such studies at ICCB-ICG, we have stage. Encouraging experiments have been niques have already yielded new insights introduced the concept of and are build- recorded, challenges have been identified, into biology. As an early example, in col- ing a new laboratory (“ICCB-Kendall and solutions are being pursued. The suc- laboration with ICCB codirector Timo- Square”) for small-molecule annotation39b cesses, however, have not yet matched the thy J. Mitchison and members of his lab, and profiling40, additional key elements distinguished history of the “ad hoc stage” we used a cytoblot screen and screening- of ChemBank. Here, the outcome of all of small-molecule explorations of life sci- by-imaging to discover monastrol—the experiments tends to be more important ence. However, as in the field of DOS, first small-molecule inhibitor of mitosis than the outcome of any individual exper- there are many reasons to feel optimistic that does not target tubulin.41a Monastrol iment. By analyzing the outcome of the 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 32 of 2,800 small molecules prepared using DOS); calmoduphilin (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).

Haptamide

Secramine Calmoduphilin Uretupamine

TetracycleTubacin Macrolide Macrocyclic biaryl

HTTP://WWW.CEN– ONLINE.ORG C&EN / MARCH 3, 2003 59 PERSPECTIVE

For chemical genetics to reach its full chemical genetic screens, the researchers tions in cancer and the possibility of re- potential, it must be embraced as a gener- have uncovered small-molecule agonists and versing their consequences through bio- al tool for exploring biology. As an approach antagonists of hedgehog signaling. Mecha- physical means.51 To explore the function to dissecting biology, like the genomic ap- nistic studies of these probes revealed that of another nucleic acid-protein interac- proach, it is not yet viewed in the same light hedgehog proteins, which act through the tion, Peter Beal and coworkers at the Uni- as biochemical or genetic approaches. Two integral membrane protein Patched, de-re- versity of Utah identified a small-molecule key challenges go hand in hand: Chemical press the G-protein coupled receptor probe of PKR, an RNA-dependent pro- genetic methods must be ac- Smoothened, which is tar- tein kinase thought to survey cells for ev- cessible, and the concepts For chemical geted by the probes. These idence of viral invasion.52 Finally, in a study behind chemical genetics studies point to the exciting that links signaling to the previously dis- must permeate the thinking genetics to possibility of endogenous, cussed cytoskeleton, fascinating insights of life scientists. Efforts to reach its full small-molecule regulators of have been gained into a signaling pathway address the former have the Smoothened proteins. that regulates actin nucleation and poly- been described throughout potential, it An entire issue of the Journal merization. Acyclic peptide53a and the small this perspective. Is the con- must be of Biology (December 2002) molecule wiskostatin53b were identified at cept of interrogating biology was recently devoted to these Harvard Medical School by Marc W. with small molecules sinking embraced as advances. Continuing with Kirschner and colleagues in chemical ge- into the mind-set of life sci- the theme of development netic screens and found to bind to the neu- entists on a broader scale a general tool and differentiation, Helene ronal-Wiskott Aldrich Syndrome Protein than in the past? Recent ev- for exploring Gilgenkrantz at the Cochin (N-WASP). Wiskostatin binding to N- idence suggests so. Institute, Paris, and cowork- WASPstabilizes an autoinhibited state and The past several years biology. ers used small-molecule di- prevents N-WASPfrom activating a down- have seen a surge both in the merizers in mice to ablate stream actin-nucleating complex (Arp2/3), number of reports of biological systems be- and regenerate, in a dose-dependent fashion, which functions to nucleate the actin poly- ing dissected with small molecules and in liver cells (hepatocytes).46 In addition, Pe- mer. The small-molecule probes revealed a the number of institutional commitments ter Schultz, at Scripps Research Institute, previously unidentified mechanism for tar- to establishing the requisite infrastructure. and coworkers in two separate studies used geting interactions between key proteins in In response to numerous queries, we have chemical genetic screens to identify small cells.53 posted on the ICCB-ICG website a how- molecules that reverse myotube formation47 to guide for building an academic screen- and that promote the differentiation of IN SUMMARY, the systematic population ing facility (Caroline Shamu at Harvard mesenchymal progenitor cells into an os- of chemical space with small molecules us- University, http://iccb.med.harvard.edu/ teoblast lineage.48 ing DOS and information science, the sys- screening/faq_hts_facility.htm).Last year, Cell signaling. Using the principle of tematic probing of biology space with these the Howard Hughes Medical Institute an- chemical genetic synthetic lethal screen- small molecules, and the sharing of results nounced its plans to develop a new research ing40, Philip Leder, Stanley J. Korsmeyer, and data using common standards and lan- campus, Janelia Farm in Leesburg, Va., with and colleagues at Harvard Medical School guage on public databases promise a future chemical genetics as one of several central screened matched cell lines differing only understanding of the relationship of chem- elements of its unique, integrated, and col- in their levels of expression of the Neu ical and biological spaces. This is an excit- laborative structure. At ICCB-ICG alone, oncogene.49 A small molecule was identi- ing and important prospect, and one that we have provided small molecules and per- fied that interfered with cell function only will require organic chemistry to play a dis- formed screens for a rapidly (and some- in cells overexpressing Neu. Mechanistic covery role. Synthetic organic chemists in what alarmingly) growing number of labo- studies revealed that the small molecule particular will need to meet the challenges ratories nationwide. Basic biological targeted the mitochondrial proton gradi- of DOS. With such an understanding and insights gained from chemical genetics ent. This is an exciting experiment both in with the availability of appropriate tools, studies from many labs worldwide signal a design and outcome. That an oncogene organic chemists may in the future design noteworthy trend in the past several years. renders cells sensitive to disruptors of synthetic pathways yielding small mole- To illustrate, I have selected several repre- mitochondrial function reveals a previous- cules targeted to the swath of chemical sentative examples from just two fields: de- ly hidden facet of cancer cell circuitry. That space optimal for modulating the swath of velopmental biology and cell signaling. mitochondria are integral components of biological space of interest. Although dif- Developmental biology. Develop- the apoptotic signaling network is well ap- ferent in its reliance on empirical observa- mental biologists uncovered the hedge- preciated. However, insights into their functions that permit “targeting,” the desired hog-signaling network, including an outline tional role in signaling were gained in early capabilities are reminiscent of those pro- of its downstream signaling elements. This 2003 when a new apoptotic signaling on- vided by natural selection, the process that pathway is involved in the development of coprotein was discovered as the target of a yields natural products. A noteworthy dif- animals and the maintenance and repair of small-molecule modulator, which was bril- ference is the anticipated time frame of the adult cells, and its disruption results in cer- liantly uncovered using another chemical former relative to the latter! tain types of cancer. The mysteries of the genetic screen.50 Downstream of these pro- pathway’s mechanistic details are deepen- teins lies the key signal integrating protein Stuart L. Schreiber is an investigator with ing. Scientists at Curis Inc.44 and in Philip p53. Using protein-binding screens, scien- the Howard Hughes Medical Institute and is A. Beachy’s lab45 at Johns Hopkins dis- tists at Pfizer identified a small molecule chair of the department of chemistry and chemi- sected the pathway with an open embrace that stabilizes the folding of mutant forms cal biology at Harvard University. He is a founder of genetic principles, yet using small mole- of DNA-binding p53; these probes are il- of the Harvard Institute of Chemistry & Cell Bi- cules as the source of perturbation. Using luminating the role of oncogenic muta- ology–Initiative for Chemical Genetics.

60 C&EN / MARCH 3, 2003 HTTP://WWW.CEN– ONLINE.ORG ENDNOTES 29. For example, see: Lee, D. et al. Org. Lett. 2 (2000): 709–712. 30. (a) Micalizio, G. C., and S. L. Schreiber. Angew. Chem. Int. Ed. 41 (2002): 1. Lander, E., commenting on the challenge of understanding cell circuitry. 152–154. (b) Micalizio, G. C., and S. L. Schreiber. Angew. Chem. Int. Ed. 2. (a) Schreiber, S. L. Chem. Eng. News, 70, No. 43 (1992): 22–32. (b) See also 41 (2002): 3272–3276. Hung, D. T. et al. Chem. Biol., 3 (1996): 623–640. 31. Pelish, H. E. et al. J. Am. Chem. Soc. 123 (2001): 6740–6741. 3. (a) Weisenberg, R. C. et al. Biochemistry 7 (1968): 4466–4479. (b) For an ex- 32. Kwon, O. et al. J. Am. Chem. Soc. 124 (2002): 13402–13404. cellent account of this exciting area of research, see also Peterson, J. R., 33. (a) Stavenger, R. A., and S. L. Schreiber. Angew. Chem. Int. Ed. 40 (2001): and T. J. Mitchison. Chem. Biol. 9 (2002): 1275–1285. 3417–3421. (b) Koehler, A., and S. L. Schreiber. Manuscript in preparation. 4. Haggarty, S. J. et al. Chem. Biol. 7 (2000): 275–286. 34. Kubota, H. et al. Chem. Biol. 9 (2002): 265–276. 5. For a review of neurotoxins, see Trends in Neuroscience, June 1996, 35. (a) Sternson, S. M. et al. Org. Lett. 3 (2001): 4239–4242. (b) Haggarty, S. J. supplement. et al. Proc. Natl. Acad. Sci. USA, in press. 6. Carlsson, A. J. Psychopharmacol. 4, No. 3 (1990): 120–126. 36. Schmidt, D. et al. J. Comb. Chem., in press. 7. Blumberg, P. M. Crit. Rev. Toxicol. 8 (1981): 199–234. 37. (a) Spring, D. R. et al. J. Am. Chem. Soc. 122 (2000): 5656–5657. (b) Spring, 8. Rosen, E. D. et al. Genes Dev. 14 (2000): 1293–1307. D. R. et al. J. Am. Chem. Soc. 124 (2002): 1354–1363. 9. Miller, D. K. et al. Nature 343 (1990): 278–281. 38. Burke, M. D., and G. Lalic. Chem. Biol. 9 (2002): 535–541. 10. Rohrer, S. P. et al. Science 282 (1998): 737–740. 39. (a) Blackwell, H. E. et al. Chem. Biol. 8 (2001): 1167–1182. (b) Clemons, P. 11. Schreiber, S. L. Science 251 (1991): 283–287. A. et al. Chem. Biol. 8 (2001): 1183–1195. (c) Blackwell, H. E. et al. Angew. 12. Belshaw, P. J. et al. Synlett (1994): 381–392. Chem. Int. Ed. 40 (2001): 3421–3425. 13. Liu, J. et al. Cell 66 (1991) 807–815. 40. Stockwell, B. R. et al. Chem. Biol. 6 (1999): 71–83. 14. Schreiber, S. L., and G. Crabtree. Harvey Society Lectures 89 (1997): 41. (a) Mayer, T. U. et al. Science 286 (1999): 971–974. (b) Peterson, R. T. et 373–380. al. Proc Natl. Acad. Sci. USA 97 (2000): 12965–12969. 15. Crabtree, G. R., and E. N. Olson. Cell 109 (2002): 867–879. 42. (a) MacBeath, G. et al. J. Am. Chem. Soc. 121 (1999): 7967–7968. (b) Her- 16. Yang, J. et al. Curr. Biol. 8 (1997): 11–18. genrother, P. J. et al. J. Am. Chem. Soc. 122 (2000): 7849–7850. (c) 17. Stockwell, B. R., and S. L. Schreiber. Curr. Biol. 8 (1998): 761–770. Barnes-Seeman, D. Angew. Chem. Int. Ed., in press. 18. Spencer, D. et al. Curr. Biol. 6 (1996): 839–848. 43. MacBeath, G., and S. L. Schreiber. Science 289 (2000): 1760–1762. 19. http://www.ariad.com. 44. Frank-Kamenetsky, M. et al. J. Biol. 1 (2002): 1:10. 20. (a) Brown, E. J. et al. Nature 369 (1994): 756–758. (b) Sabatini, D. M. et al. 45. Chen, J. K. et al. Proc. Natl. Acad. Sci. USA 99 (2002): 14071–14076. Cell 78 (1994): 34–43. 46. Mallet, V. O. et al. Nat. Biotechnol. 20 (2002): 1234–1239. 21. Shamji, A. F. et al. Curr. Biol. 10 (2000): 1574–1581. 47. (a) Rosania, G. R. et al. Nat. Biotechnol. 18 (2000): 304. (b) For commen- 22. Kuruvilla, F. G. et al. Nature 416 (2002): 653–657. tary, see: Frederickson, R. Nat. Biotechnol. 18 (2000): 250. 23. Taunton, J. et al. Science 272 (1996): 408–411. 48. Wu, X. et al. J. Am. Chem. Soc. 124 (2002): 14520–14521. 24. Brownell, J. E. Cell 84 (1996): 843–851. 49. Fantin, V. R. et al. Cancer Cell 2 (2002): 29–42. 25. Schreiber, S. L., and B. E. Bernstein. Cell 111 (2002): 771–778. 50. (a) Jiang, X. et al. Science 299 (2003): 223–226. (b) Nicholson, D. W., and 26. Schreiber, S. L. Science 287 (2000): 1964–1969. N. A. Thornberry. Science 299 (2003): 214–215. 27. (a) Baum, R. Chem. Eng. News 76 No. 46 (1998): 31–34. (b) Gura, T. Nature 51. Foster, B. A. et al. Science 286 (1999): 2507–2510. 407 (2000): 282–284, (c) For a review of ICCB-ICG, see: http://iccb.med. 52. Carlson, C. B. et al. ChemBioChem 3 (2002): 859–865. harvard. edu. 53. (a) Peterson, J. R. et al. Proc. Natl. Acad. Sci. USA 98 (2001): 10624–10629. 28. Adam, D. Nature 411 (2001): 873. (b) Peterson, J. R. et al. Unpublished results and ref. 3(b).

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