DOI: 10.1126/science.1153813
`, 1536 (2008); 319Science
` et al.David T. George,
`Therapy for Alcoholism
`Neurokinin 1 Receptor Antagonism as a Possible
`www.sciencemag.org (this information is current as of March 14, 2008 ):
`The following resources related to this article are available online at
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` http://www.sciencemag.org/cgi/content/full/319/5869/1536#otherarticles
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`28 November 2007; accepted 5 February 2008
`10.1126/science.1153498
`Neurokinin 1 Receptor Antagonism
`as a Possible Therapy for Alcoholism
`David T. George,
`1* Jodi Gilman,1* Jacqueline Hersh,1* Annika Thorsell,1*
`David Herion,1 Christopher Geyer,2 Xiaomei Peng,3 William Kielbasa,3 Robert Rawlings,1
`John E. Brandt,3 Donald R. Gehlert,3 Johannes T. Tauscher,3 Stephen P. Hunt,4
`Daniel Hommer,1 Markus Heilig1†
`Alcohol dependence is a major public health challenge in need of new treatments. As alcoholism
`evolves, stress systems in the brain play an increasing role in motivating continued alcohol use
`and relapse. We investigated the role of the neurokinin 1 receptor (NK1R), a mediator
`of behavioral stress responses, in alcohol dependence and treatment. In preclinical studies, mice
`genetically deficient in NK1R showed a marked decrease in voluntary alcohol consumption and
`had an increased sensitivity to the sedative effects of alcohol. In a randomized controlled
`experimental study, we treated recently detoxified alcoholic inpatients with an NK1R antagonist
`(LY686017; n = 25) or placebo (n= 25). LY686017 suppressed spontaneous alcohol cravings,
`improved overall well-being, blunted cravings induced by a challenge procedure, and attenuated
`concomitant cortisol responses. Brain functional magnetic resonance imaging responses to
`affective stimuli likewise suggested beneficial LY686017 effects. Thus, as assessed by these
`surrogate markers of efficacy, NK1R antagonism warrants further investigation as a treatment
`in alcoholism.
`A
`lcohol use accounts for 4% of global
`disease burden (1). Alcohol dependence,
`or alcoholism, is characterized by a chron-
`ic relapsing course, in which alcohol-associated
`cues and stress are known relapse triggers (2–6).
`Recent research suggests that neural systems
`mediating behavioral stress responses may offer
`useful targets for pharmacotherapy of alcohol-
`ism. In animal models, excessive alcohol con-
`sumption that results from a history of alcohol
`dependence is accompanied by increased be-
`havioral sensitivity to stress (7). Up-regulated
`corticotropin-releasing hormone (CRH) signal-
`ing in extrahypothalamic brain sites contributes
`to these dependence-induced changes, but other
`stress-related neurotransmitters may also play
`a role.
`One such neurotransmitter is substance P
`(SP), which together with its preferred neuro-
`kinin 1 receptor (NK1R) is highly expressed in
`brain areas involved in stress responses and
`drug reward, including the hypothalamus, amyg-
`dala, and nucleus accumbens. In rodents, psy-
`chological stressors induce release of SP in the
`amygdala, whereas genetic deletion or pharma-
`cological blockade of NK1R inhibits the asso-
`ciated behavioral responses (8). Furthermore,
`genetic deletion of NK1Rs causes a loss of con-
`ditioned place preference for opiates and opiate
`self-administration (9, 10). In humans, the NK1
`antagonist GR205171 reduces symptoms of so-
`cial anxiety and suppresses brain responses to the
`Trier Social Stress Test (TSST) (11). Together,
`these findings suggest that blockade of NK1Rs
`might modulate stress- and reward-related pro-
`cesses of importance for excessive alcohol use
`and relapse. To our knowledge, no data are pres-
`ently available to address this hypothesis.
`We first explored preclinically whether in-
`activation of NK1R might modulate stress- and
`reward-related processes that impact alcohol
`use. We chose a genetic inactivation strategy,
`because available NK1R antagonists have lim-
`ited activity in rats and mice, because of insuf-
`ficient NK1R amino acid homology between
`humans and these rodent species (8 ). We
`evaluated NK1R null-mutant mice for voluntary
`alcohol consumption, alcohol sensitivity, and
`alcohol metabolism (12). NK1R null mice (13)
`were back-crossed into a C57BL/6 background
`for 10 generations to ensure that there was ade-
`quate voluntary alcohol consumption in control
`animals (14). We used a two-bottle free-choice
`model with increasing alcohol concentration,
`and alcohol was continuously available. Wild-
`type littermates (+/+) ultimately consumed in
`excess of 10 g alcohol/kg of body weight per
`day at the end of an escalation procedure in which
`alcohol concentration was gradually increased
`from 3 to 15% over 60 days.
`Alcohol consumption by NK1R
`–/– mice was
`markedly lower than that by wild-type controls
`(Fig. 1A). The difference was most prominent at
`higher alcohol concentrations, at which con-
`sumption motivated by pharmacological alcohol
`effects dominates over intake for taste, calories,
`or other nonpharmacological effects (14). Alco-
`hol consumption by heterozygous (+/–)m i c e
`was similar to wild-type controls, highlighting
`the necessity for near-complete inactivation of
`NK1Rs to suppress alcohol consumption. Sev-
`1Laboratory of Clinical and Translational Studies, National
`Institute on Alcohol Abuse and Alcoholism, National Institutes
`of Health, Bethesda, MD 20892, USA.2Department of Nurs-
`ing, Mark O. Hatfield Clinical Research Center, National
`Institutes of Health, Bethesda, MD 20892, USA.
`3Lilly Re-
`search Laboratories, Indianapolis, IN 46285, USA.4Depart-
`ment of Anatomy and Developmental Biology, University
`College London, London WC1E 6BT, UK.
`*These authors contributed equally.
`†To whom correspondence should be addressed. E-mail:
`markus.heilig@mail.nih.gov
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`eral observations indicated that the reduction in
`alcohol intake was specific. Thus, relative pref-
`erence for alcohol was also markedly reduced
`[(F2,45 = 13.6, P < 0.0001), whereas water in-
`take was unaffected by genotype (F2,45 = 0.54,
`P = 0.71)]. Additional control experiments indi-
`cated that the reduction in alcohol intake was not
`caused by altered thirst or taste preference (12).
`Alcohol sensitivity, measured as the time
`required to regain the righting reflex after a high
`dose of alcohol (3.5 g/kg), was markedly in-
`creased in NK1
`–/– mice (Fig. 1B). This profile is
`consistent with an abundance of animal data show-
`ing an inverse relation between alcohol sensitivity
`and motivation to consume alcohol, as well as
`human findings of an inverse relation between
`alcohol sensitivity and alcoholism risk (15).
`To assess the potential clinical relevance of
`these results, we conducted an experimental study
`in human alcohol-dependent patients (fig. S1
`and table S1), in which we evaluated the effects
`of NK1R antagonism on processes related to
`relapse. We studied LY686017 (16), a high-affinity,
`selective NK1R antagonist that is orally available
`and brain penetrant (Fig. 2A). Preclinical phar-
`macology, safety, and human pharmacokinetics
`of LY686017 will be reported separately. Activ-
`ity of LY686017 to reduce drinking was not as-
`sessed in preclinical experiments because, similar
`to other human NK1R antagonists, LY686017
`has insufficient affinity for the mouse or rat
`NK1R. LY686017’s brain penetrance and NK1R
`occupancy were established in a human positron
`emission tomography (PET) study of eight healthy
`volunteers (12). On the basis of these results,
`we used a dose of 50 mg daily, which yields a
`>90% blockade of central NK1R. LY686017 at
`this dose was well tolerated (table S2) (12), in
`agreement with prior reports indicating that
`NK1 antagonists as a class are safe and well
`tolerated (8).
`Given the role of SP and NK1 receptors in
`stress and anxiety responses, we targeted subjects
`with high trait anxiety. Participants (25 per arm;
`age: 21 to 65 years) had a diagnosis of alcohol
`dependence, alcohol problems as the primary
`complaint, alcohol use within the last month,
`and >39 on the Spielberger Trait Anxiety In-
`ventory (STAI) (17). They were hospitalized
`throughout the study and had completed with-
`drawal treatment before entering the study if
`needed. For details, see (12).
`We found that LY686017 suppressed spon-
`taneous alcohol cravings, as measured by the
`Alcohol Urge Questionnaire (AUQ) (Fig. 2B).
`As expected, these cravings declined over time
`in the protected inpatient environment and were
`minimal in the majority of patients by the end of
`the 4-week study period. However, overall, the
`cravings were sufficiently intense to allow de-
`tection of the medication effect. Weekly ratings
`by a blinded observer likewise suggested that
`LY686017 had a beneficial effect on global im-
`provement and severity measured by the Clini-
`cian’s Global Impression (CGI) scale (Fig. 2B).
`In contrast, twice-weekly ratings collected to ob-
`tain measures of general psychopathology showed
`no treatment effects on anxious or depressive
`psychopathology (F
`1,102 =0 . 7 ,P = 0.40). This
`suggests that the improvements observed might be
`specific for brain processes related to alcoholism.
`We further measured craving responses to a
`combined stress (TSST) and alcohol-cue challenge
`(12) and found that treatment with LY686017
`reduced the resulting AUQ craving response (Fig.
`3A). Treatment with LY686017 also suppressed
`the concomitant cortisol response to the challenge
`(Fig. 3B). A study using PET has previously
`shown that GR205171, another NK1 antagonist,
`suppresses amygdala activation in response to
`the TSST in social phobics, but in that study,
`NK1R antagonism failed to produce effects on
`subjective, self-reported measures in response to
`the challenge (11). This shows that subjective re-
`sponses to a challenge are transient, and their
`detection is critically dependent on the time point
`and the assessment instrument chosen. The fact
`that we detected consistent effects of LY686017
`on both subjective and neuroendocrine responses
`to the craving challenge, therefore, supports the
`robustness of the LY686017 effect.
`Fig. 1.Voluntary alcohol intake and alcohol sensitivity in NK1 null (–/–), heterozygous (+/–) and wild-
`type (+/+)m i c e .(A) Voluntary consumption in a two-bottle free-choice paradigm with continuous
`alcohol access. Alcohol was introduced as a 3% solution in water, and concentration was escalated over
`time, as indicated above the graph, to avoid taste aversion and to achieve pharmacologically active
`levels of alcohol consumption. The other bottle contained water. Data are means ± SEM;n =1 6p e r
`group. The +/– group is omitted for clarity; its alcohol consumption was virtually identical to that of +/+
`mice. There was a main genotype effect (F2,40 = 929.6,P < 0.00001). On Tukey’s post hoc test,–/–
`mice differed from both +/+ and +/– mice (both:P < 0.001); the latter two genotypes were virtually
`identical (P =0 . 9 2 ) .(B) Alcohol sensitivity in mice, measured as the time to regain the righting reflex
`after a 3.5-g/kg dose of alcohol. Data are means ± SEM;n = 12 to 17/group). Time to regain the
`righting reflex was significantly longer for the NK1–/– mice as compared with their wild-type controls
`(F2,42 =7 . 8 ,P = 0.0014; Tukey’sp o s th o cP =0 . 0 0 1–/– vs. +/+).
`Fig. 2. Effect of the NK1R antagonist
`LY686017 on spontaneous cravings and glob-
`al well-being in hospitalized alcoholics. (A)
`LY686017 (2-chloro-phenyl)-{2-[5-pyridin-
`4-yl-1-(3,5-bistrifluoromethyl-benzyl)-1H-
`[1,2,3]triazol-4-yl]-pyridin-3-yl}-methanone)
`(16), the selective, brain-penetrant NK1R
`antagonist used in this study. (B)C h a n g e
`from baseline for spontaneous alcohol crav-
`ings, as measured by twice-weekly ratings
`using the AUQ. The first value reflects the
`baseline rating obtained during the placebo lead-in week; the following ratings
`are from the active treatment phase. Data are means ± SEM. Baseline values
`for placebo and LY686017 groups were 19.1 ± 2.4 and 19.7 ± 2.3, respectively.
`Controlling for pretreatment baseline and sex, there was a highly significant
`decline of cravings over time (F
`5,220=3 7 . 0 5 ,P < 0.0001), and a significant main
`effect of treatment (F1,100 =4 . 4 ,P =0 . 0 3 9 )o nt h i so u t c o m e .(C) Change from
`baseline on weekly observer-based ratings using the Severity scale of the CGI
`rating questionnaire. The first value reflects the baseline rating obtained during
`the placebo lead-in week; the following ratings are from the active phase.
`Average baseline ratings for the placebo and LY686017 groups were 3.80 ± 0.15
`and 3.88 ± 0.13, respectively. Data are means ± SEM. Controlling for
`pretreatment baseline, sex, and body mass index, there was a significant effect
`of treatment (F
`1,42 =1 1 . 9 ,P = 0.001). Very similar results were obtained on the
`improvement scale of the CGI (main treatment effect:F1,44 =8 . 4 ,P = 0.006).
`N
`N
`N
`N
`N
`O
`Cl
`F FF
`F
`F
`F
`AB C
`www.sciencemag.org SCIENCE VOL 319 14 MARCH 2008 1537
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`Finally, we investigated whether LY686017
`affected brain responses to standardized affec-
`tive stimuli, with a pattern suggestive of bene-
`ficial effects on relapse-related mechanisms.
`We measured blood oxygen–level–dependent
`(BOLD) activity using functional magnetic
`resonance imaging (fMRI), following presenta-
`tion of negative and positive emotional stimuli
`from the International Affective Picture System
`(IAPS) (18) and pictures of alcoholic or neutral
`beverages (12). Alcoholics exhibit exaggerated
`behavioral and brain responses to images asso-
`ciated with negative affect, and conversely, exhib-
`it reduced brain responses to standard positive
`images (19). In agreement with these earlier
`observations, we found that placebo-treated al-
`coholics showed robust brain responses to
`negative affective images. Alcoholics who re-
`ceived LY686017 had less activation to the
`negative images than the placebo group in
`several brain regions associated with emotional
`response to visual stimuli [Fig. 4 and table S3
`(12)]. In particular, the LY686017 group had
`less activation in the insula, a brain region
`whose activation correlates with subjective mea-
`sures of craving (20), and which has recently
`been implicated in the maintenance of addictive
`behavior (21). In addition, the LY686017-treated
`group showed greater brain activation to the
`positive IAPS images than the placebo-treated
`group (Fig. 4). This may reflect an overall shift
`in the balance between positive and negative
`emotionality as indicated by the improvement
`detected on the CGI. A recent report suggests
`that greater activation to positive images in the
`striatum and thalamus of treated alcoholics pre-
`dicts less alcohol consumption over the next 6
`months (22).
`Basic neuroscience research has identified
`numerous candidate targets for pharmacological
`treatment of alcoholism (4), but translation into
`clinical development has been limited. Surrogate
`markers of efficacy that can be evaluated in al-
`coholics under safe, closely monitored condi-
`tions can facilitate translation. In this context,
`self-reported cravings are of interest, because
`they are triggered in humans by the same types
`of stimuli that, in animal models, induce relapse
`to alcohol-seeking (3, 5, 6). Human data also
`show that cravings correlate with clinical out-
`comes (23) and are sensitive to a clinically ef-
`fective alcoholism medication, naltrexone (24).
`For these reasons, we chose spontaneous as well
`as challenge-induced cravings as primary out-
`comes in our study. Both these outcomes were
`beneficially affected by the NK1R antagonist.
`The concept of craving and its ability to pre-
`dict clinical outcomes has also invited some
`debate (25). Although effects on cravings and
`clinical outcomes do correlate in the case of
`naltrexone, it is unknown whether this relation
`will hold up for other pharmacodynamic mech-
`anisms. This will only be possible to establish
`once additional compounds with clinical efficacy
`are identified. Until then, a translational approach
`that is guided by animal data and combines
`craving measures with a profile across a broader
`range of experimental outcomes appears to offer
`an attractive approach to drug development for
`alcoholism. Using this approach, we provide
`here consistent data across a range of measures
`suggesting that NK1R antagonism might be of
`therapeutic value in alcoholism. A possible mech-
`anism for this is suppression of pathologically
`elevated amygdala activity thought to develop
`following a history of alcohol dependence (7).
`Our results were obtained in anxious alco-
`holics. Larger trials, stratified for anxiety mea-
`sures, will be required to address whether the
`effect of NK1R antagonism is specific for this
`population.
`Other NK1R antagonists have previously
`been tested clinically as a therapy for major de-
`pression (8, 26), but the results were inconsistent,
`and development of these drugs was stopped.
`Additional studies will be required to determine
`whether development of NK1R antagonists for
`alcoholism will be more successful, but recent
`findings with another stress-related neuropeptide
`system, CRH, suggest that this may be the case.
`Thus, analysis in animal models of alcoholism
`shows that the CRH system is quiescent under
`physiological or near-physiological conditions,
`and under these conditions, no activity of CRH
`antagonists is found. The CRH system is, how-
`ever, pathologically activated following a history
`of alcohol dependence, revealing activity of CRH
`antagonists (7). These findings are in agreement
`with a general principle proposed for neuropep-
`Fig. 3.Effect of the NK1R
`antagonist LY686017 on
`subjective craving and
`neuroendocrine response
`to a challenge session in
`hospitalized alcoholics. (A)
`Subjective craving ratings
`on the AUQ. Data are
`means ± SEM. Possible
`score range is 8-56. Con-
`trolling for sex and age,
`there was a robust craving response to the challenge on the AUQ, as shown by an effect of the repeated
`measures (time) factor (F
`3,129 =4 . 8 ;P = 0.003), and a highly significant post hoc difference of both the
`poststress and the post–alcohol cue value versus baseline (Dunnett’st e s t ,P <0 . 0 0 1f o re a c h
`comparison); the recovery rating was back to baseline level. A modulation of the challenge response by
`treatment was shown as a time × treatment interaction (F
`3,129 =5 . 2 2 ;P = 0.002). Responses were also
`obtained on a simple visual analog scale (VAS). Although this was less sensitive, a similar effect was
`suggested at a trend level (time × treatment:F4,176 = 2.10;P = 0.08). There was also a robust anxiety
`response (F3,132 =4 . 1 ,P = 0.008), but no treatment or interaction effect on this variable. (B)
`Neuroendocrine response. Data are means ± SEM; arrow points to start of challenge. Controlling for
`sex, there was a robust cortisol response to the combined stress and cue exposure, as shown by a highly
`significant effect of the repeated measures (time) factor (F
`8,344 = 12.9, P << 0.0001). A significant
`differential response to the combined stress and cue exposure as a function of treatment was
`demonstrated by the interaction of time × treatment (F8,344 =2 . 6 ,P =0 . 0 1 0 ) .
`Fig. 4.Effects of LY686017
`on fMRI BOLD responses to
`visual affective stimuli in
`hospitalized alcoholics. In
`the placebo group, there
`were robust activations to
`the negative stimuli in the
`inferior frontal gyrus, insula,
`and middle temporal gyrus;
`the LY686017-treated group
`had significantly less activa-
`tion in these areas. The pla-
`cebo group had very little
`activation in response to the
`p o s i t i v ee m o t i o n a ls t i m u l i ;t h e
`LY686017-treated group had
`greater activation in the tha-
`lamus, caudate (including
`ventral putamen), lingual
`gyrus, and several temporal
`areas. Group statistical maps
`are superimposed upon a T1
`structural image in Talairach
`space. See table S2 (12) for details.
`Placebo group
`Response to Negative Images
`p < 0.001 p < 0.01
`Inferior frontal
`gyr
`us/insula
`Middle
`temporal gyrus
`Nucleus accumbens/
`putamen
`Response to Positive Images
`L Y686017-treated group
`14 MARCH 2008 VOL 319 SCIENCE www.sciencemag.org1538
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`tide systems (27). Along these lines, activation
`of the SP-NK1R system may not be a consistent
`feature of depressive illness. If, however, a path-
`ological activation of the SP-NK1R system fol-
`lows a history of alcohol dependence, similar to
`CRH, NK1 antagonism may have a considera-
`ble potential as a treatment for alcoholism.
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`28. We thank nurse manager J. Johnson and her treatment
`staff at the 1SE Unit of the NIH Clinical Center, as well
`as D. Hill, L. Doty, and C. Jones for their invaluable
`contributions to this study. R. Anton, S. Thomas,
`R. Miranda, and P. Monti are gratefully acknowledged for
`input in the design stages of the clinical study. Supported
`by the intramural research budget of the National
`Institute on Alcohol Abuse and Alcoholism, NIH. This
`work was carried out under a Collaborative Research and
`Development Agreement (CRADA) between the U.S.
`Government and Eli Lilly and Co. All coauthors so
`indicated are employees of Eli Lilly and Co.; the
`remaining authors have no conflict of interest to disclose.
`Supporting Online Material
`www.sciencemag.org/cgi/content/full/1153813/DC1
`Materials and Methods
`Fig. S1
`Tables S1 to S3
`References
`5 December 2007; accepted 6 February 2008
`Published online 14 February 2008;
`10.1126/science.1153813
`Include this information when citing this paper.
`Using Engineered Scaffold
`Interactions to Reshape MAP Kinase
`Pathway Signaling Dynamics
`Caleb J. Bashor,
`1,2 Noah C. Helman,1 Shude Yan,1 Wendell A. Lim1*
`Scaffold proteins link signaling molecules into linear pathways by physically assembling them into
`complexes. Scaffolds may also have a higher-order role as signal-processing hubs, serving as the
`target of feedback loops that optimize signaling amplitude and timing. We demonstrate that the
`Ste5 scaffold protein can be used as a platform to systematically reshape output of the yeast
`mating MAP kinase pathway. We constructed synthetic positive- and negative-feedback loops by
`dynamically regulating recruitment of pathway modulators to an artificial binding site on Ste5.
`These engineered circuits yielded diverse behaviors: ultrasensitive dose response, accelerated or
`delayed response times, and tunable adaptation. Protein scaffolds provide a flexible platform for
`reprogramming cellular responses and could be exploited to engineer cells with novel therapeutic
`and biotechnological functions.
`I
`n cells, signaling proteins that make up a
`pathway are often physically organized into
`complexes by scaffold proteins (1–3). Scaf-
`folds direct information flow; they promote sig-
`naling between proper protein partners and
`prevent improper cross talk. Scaffolds may also
`play a role in shaping the quantitative response
`behavior of a pathway. The scaffold complex
`could serve as a central hub for feedback loops
`that modulate the recruitment or activity of
`pathway members on the scaffold. Such feedback
`loops could tune pathway dose response and
`dynamics— the change in output over time. Quan-
`titative response behavior is critical for signaling;
`the behavior of a pathway must match its specific
`physiological function (4). Scaffolds may there-
`fore provide a platform for evolutionarily tuning
`response behaviors for optimal fitness (5, 6).
`We used a synthetic biology approach to
`explore this hypothesis; we tested whether a
`scaffold protein can be used as a platform for
`engineering synthetic feedback loops and wheth-
`er these loops can be used to systematically
`reshape pathway response behavior (7). We used
`the yeast mating mitogen-activated protein
`(MAP) kinase pathway as a model system, be-
`cause it is highly tractable for pathway engineer-
`ing. First, proper connectivity of this pathway is
`dependent on the scaffold protein Ste5, which
`binds the three core kinases— Ste11 (a MAP ki-
`nase kinase kinase or MAPKKK), Ste7 (a MAP
`kinase kinase, or MAPKK), and Fus3 (a MAP
`kinase, or MAPK)— that successively phospho-
`rylate and activate one another (Fig. 1A) (8, 9).
`The critical role in determining pathway con-
`nectivity is highlighted by the observation that
`chimeric scaffolds can be used to redirect path-
`way input and output linkages (10, 11). Second,
`MAP kinase pathways appear to be functionally
`plastic; they are found in all eukaryotic species,
`but in individual cases display widely varied be-
`haviors. For example, the yeast mating pathway
`shows a largely linear transcriptional response
`(12–14), whereas theXenopusoocyte maturation
`pathway displays a switchlike dose response (15).
`MAPK pathways also show diverse dynamic
`behavior; some yield a sustained response to
`stimulation, whereas others show a pulselike
`transient response. These distinct pathway dy-
`namics are critical for determining physiological
`output (16–21).
`Our goal was to overlay the endogenous
`mating pathway with synthetic feedback loops in
`order to systematically alter its response to mat-
`ing pheromone (a-factor) stimulation. A simple
`way to construct a synthetic feedback loop would
`be to dynamically recruit pathway modulators to
`the scaffold in a manner that is dependent on
`pathway output. We first tested whether con-
`stitutive recruitment of modulator proteins could
`alter pathway flux. We created a new recruitment
`site on Ste5 by fusing a leucine zipper hetero-
`dimerzation module (22) to its C terminus. Mod-
`ulator proteins fused to complementary zippers
`were expressed and recruited to the scaffold (Fig.
`1B). Two pathway modulators were recruited:
`1Department of Cellular and Molecular Pharmacology,
`University of California at San Francisco, 600 16th Street,
`San Francisco, CA 94158, USA.2Graduate Group in Biophysics,
`University of California at San Francisco, 600 16th Street, San
`Francisco, CA 94158, USA.
`*To whom correspondence should be addressed. E-mail:
`lim@cmp.ucsf.edu
`www.sciencemag.org SCIENCE VOL 319 14 MARCH 2008 1539
`REPORTS
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