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THE SMALL-MOLECULE APPROACH TO BIOLOGY

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THE SMALL-MOLECULE APPROACH TO BIOLOGY Powered By Docstoc
					IMAGE BY CATHY NEWTON AND ERIC KELLER, HOWARD HUGHES MEDICAL INSTITUTE
                                                                                                                            PERSPECTIVE




                                                                         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-mole-
                                                                                                                                                            cule and genetic approaches have increased
                                                                                                                                                            the generality of the small-molecule ap-




                                                         S
                                                                                      mall molecules have long been associated
                                                                                                                                                            proach. 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 com-
                                                                                                                                                            mon for a life scientist to ask, “Should I
                                                                         as 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-

                                                                         H T T P : / / W W W. C E N – O N L I N E . O R G                                                 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                                                                                      H T T P : / / W W W. C E N – O N L I N E . O R G
          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
   PPAR 8; 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 FLAP and
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

H T T P : / / W W W. C E N – O N L I N E . O R G                                                                  C&EN / MARCH 3, 2003        53
                                                            PERSPECTIVE

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-                 T or2p, 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                                                                                   H T T P : / / W W W. C E N – O N L I N E . O R G
                                                                                                                                          SIGNALING NETWORKS Small-molecule-
IMAGE COURTESY OF STUART SCHREIBER, HARVARD UNIVERSITY




                                                                                                                                          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                                                         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
                                                                         Extracellular signal leads          Nuclear signal leads
                                                                                                                                          the cytoplasm and nucleus of cells. When the
                                                                         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
                                                                                                                                          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 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       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: “ I be-
                                                                                              Am            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-

                                                         H T T P : / / W W W. C E N – O N L I N E . O R G                                                                     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, alde-
 hydes 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         A primary 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).

56   C&EN / MARCH 3, 2003                                                                                  H T T P : / / W W W. C E N – O N L I N E . O R G
                                                            PERSPECTIVE

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-
ers of products having different skeletons.
                                                  example, a Gaussian dis-
                                                  tribution, or an even dis-
                                                                               The goals are to                     an ontology that will al-
                                                                                                                    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-
sulting in a variety of skeletally distinct,
                                                  planning of DOS. In order
                                                  to facilitate the planning of
                                                                               found to overlap                     only necessities being a
                                                                                                                    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-                                            only have to turn to our
actions of a variety of electrophilic reagents,   rithms that identify the
                                                                               space that                           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. Y  oung 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                                                                                    H T T P : / / W W W. C E N – O N L I N E . O R G
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
   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).




                                                                                                Haptamide




                         Secramine                       Calmoduphilin                                                Uretupamine




                   Tetracycle                         Tubacin                               Macrolide                  Macrocyclic biaryl



H T T P : / / W W W. C E N – O N L I N E . O R G                                                                      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. T     wo    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
address the former have
                                  reach its full                  small-molecule regulators of
                                                                  the Smoothened proteins.
                                                                                                   have been gained into a signaling pathway
                                                                                                   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-                                     of Biology (December 2002)       molecule wiskostatin53b were identified at
cept of interrogating biology
                                  must be                         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 func-    tions 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                                                                                      H T T P : / / W W W. C E N – O N L I N E . O R G
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H T T P : / / W W W. C E N – O N L I N E . O R G                                                                                     C&EN / MARCH 3, 2003             61

				
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