to explore the effects of colloidal aggregation on gpcr activity , 
 we used four molecules well - characterized for colloid formation at 
 micromolar concentrations : clotrimazole , itraconazole , quercetin , 
 and tipt ( figure 1 ) . we used the 
 chemokine receptors ccr4 and cx3cr1 , and the vasopressin 2 receptor 
 ( v2r ) as model peptide - activated gpcrs , measuring activity 
 using the -arrestin recruitment - mediated luciferase reporter 
 assay that quantifies receptor activation / inhibition upon ligand binding . as the four organic molecules bear little physical 
 or topological similarity to known ligands of these gpcrs , we expected 
 soluble , monomeric forms of the organic molecules to be relatively 
 inert to these targets . thus , broad gpcr activity would , in itself , 
 be an indication of colloid formation . aggregating small molecules 
 used in this study . our first question was 
 thus whether these known aggregators have 
 gpcr activity at concentrations above their known cac values . quercetin , itraconazole , and tipt inhibited ccl22-dependent ccr4 
 activation with ic50 values of 10.2 m , 0.5 m , 
 and 3.5 m , respectively ( table 1 ) . similarly , 
 all four of the compounds inhibited cx3cr1 activity after stimulation 
 with cx3cl1 ; ic50 values were 2.0 m for clotrimazole , 
 0.1 m for itraconazole , 4.5 m for quercetin , and 2.2 
 m for tipt ( table 1 ) . last , clotrimazole , 
 itraconazole , quercetin , and tipt also inhibited vasopressin - dependent 
 activity of v2r , with ic50 values of 2.7 m , 
 0.8 m , 7.0 m , and 0.4 m , respectively ( table 1 ) . these ic50 values are within the range 
 where colloidal inhibition has been observed for these compounds . this promiscuous inhibition of 
 various gpcr proteins by molecules unrelated to known target ligands , 
 at concentrations at which these molecules have been observed to form 
 colloids , suggests that the four aggregating compounds may be acting 
 as nonspecific colloidal inhibitors of the three gpcrs . meanwhile , 
 in nontransfected controls the colloids had no measurable nonspecific 
 effects on cell viability ( si figure s2 ) . . the pic50 and corresponding 
 ic50 values on direct treatment with aggregators , after 
 centrifugation , and with detergent are shown . it has been shown that colloids can be removed from solution 
 by centrifugation . to test whether the colloidal particles were themselves the inhibitory 
 species , we centrifuged aqueous mixtures of the colloids , and treated 
 the cells with the resulting supernatant . if colloids are indeed the 
 inhibitory species , rather than the soluble monomer , the supernatant 
 should show less activity than the original mixture . alternatively , 
 solutions of well - behaved inhibitors are unaffected by such centrifugation . consistent with a colloidal mechanism , the inhibitory potency of all 
 four inhibitors was substantially weakened by centrifugation , relative 
 to the noncentrifuged control ( figure 2 ) . against 
 ccr4 , itraconazole showed a 36-fold increase of its ic50 ; changes in inhibitory activity were also pronounced for quercetin 
 ( table 1 ) . against cx3cr1 , clotrimazole showed 
 an increase in ic50 , from 2.0 to 10.8 m , while the 
 shift for itraconazole was much more substantial , increasing from 
 0.1 to 5.0 m ( table 1 ) . similarly , clotrimazole , 
 itraconazole , and quercetin all showed substantial ic50 increases against the v2r after centrifugation : from 
 2.7 to 15.1 m , 0.8 to 11.9 m , and 7.0 to 37.8 m , 
 respectively ( table 1 , figure 2 ) . these results are consistent with a colloidal - based mechanism , 
 given that removal of colloidal particles by centrifugation reduced 
 the inhibition caused by these molecules . concentration  response curves 
 are shown for ccr4 , cx3cr1 , and v2r in the -arrestin 
 recruitment assay . circles represent data points for aggregators without 
 treatment ; squares represent data points after centrifugation ( sem ) . 
 calculated pic50 and ic50 values are displayed 
 in table 1 . although the addition of nonionic detergents can disrupt 
 colloid 
 formation , the use of detergents in cell 
 culture often has toxic effects , and this had long been a stumbling 
 block in investigating colloidal effects on cell - based assays . however , 
 recent work has shown that low concentrations of tween-80 are both 
 well - tolerated in cell culture and disrupt many colloids . to investigate the ability of tween-80 to disrupt 
 the colloids formed by clotrimazole , itraconazole , quercetin , and 
 tipt , we measured particle formation in the presence and absence of 
 this detergent by dynamic light scattering ( dls ) . we found that tween-80 
 completely disrupts the colloids formed by these small molecule aggregators 
 ( figure 3 ) . particle formation by colloid - forming 
 molecules measured by dls 
 in the ( a ) absence of tween-80 and ( b ) presence of tween-80 . in live cell - based control experiments , 
 the percentage of tween-80 
 tested had little or no measurable effect on the growth of the cells , 
 nor on the activity of the receptors . we note that because the tween-80 
 is removed with the rest of the cell media before measuring -arrestin 
 signaling , via the induction of luciferase , there is no detergent 
 effect on luciferin itself or luciferase activity . knowing that tween-80 
 disperses colloids but does not affect the assay itself again , consistent with a colloidal mechanism , the addition of detergent 
 substantially diminished the activity of all four inhibitors against 
 the three receptors , from 3- to 200-fold , when compared to inhibition 
 in the absence of detergent ( figure 4 ) . against 
 ccr4 , on addition of tween-80 the ic50 values of itraconazole 
 and quercetin increased from 0.5 to 11.4 m and from 10.2 to 
 > 100 m , respectively , while tipt s shift was more 
 moderate , 
 increasing from 3.5 to 9.9 m ( table 1 ) . against cx3cr1 , all four compounds showed substantial increases 
 in ic50 values on addition of tween-80 : 2.0 to 7.9 m 
 for clotrimazole ; 0.1 to 20.8 m for itraconazole ; 4.5 to > 32 
 m for quercetin ; and from 2.2 to > 32 m for tipt ( table 1 ) . similarly , the inhibition of v2r decreased 
 substantially in the presence of detergent : the ic50 for 
 itraconazole increased from 0.8 to 6.0 m ; tipt increased from 
 0.4 to 45.0 m ; and the ic50 for quercetin doubled , 
 from 7.0 to 15.2 m ( table 1 ) . the ic50 values calculated for all four aggregators against the gpcrs 
 measured here are similar to the ic50 values observed for 
 the same molecules against soluble enzymes that have become standard 
 markers for aggregation , such as -lactamase and cruzain . response 
 curves are shown 
 for ccr4 , cx3cr1 , and v2r in the -arrestin recruitment 
 assay . circles represent data points for aggregators without treatment ; 
 squares represent data points after addition of detergent ( sem ) . 
 calculated pic50 and ic50 values are displayed 
 in table 1 . we also investigated the inhibition of v2r using 
 a second 
 assay type , ca mobilization . similar ic50 values 
 were observed as with the -arrestin recruitment assays for 
 all four aggregators ( si table s1 , figure s1 ) . intriguingly , the dose  response curves were steep in the 
 ca mobilization assay , with hill slope coefficients as 
 high as 5 ( si table s1 ) ; both observations 
 are consistent with a colloidal mechanism . we note that , in this ca mobilization assay , inhibition was unaffected by centrifugation 
 or detergent treatment . this likely reflects the composition of the 
 ( proprietary ) calcium assay dye kits , which contain fluorescent dyes 
 as well as masking dyes . many such dyes are well - known aggregators , and have been shown to interact with each other and 
 other aggregators in nonadditive ways , substantially altering colloidal characteristics and assay results . consistent with this view , the assay kit alone formed colloid - like 
 particles by dls of 166 nm in size , and inhibited two unrelated enzymes , 
 chymotrypsin and malate dehydrogenase ( si table 
 s2 ) . as the fluo-4 direct kit composition is proprietary , these 
 effects can not be further disentangled . for the purposes of this study , the key result is that ic50 values measured with the four 
 aggregators by calcium mobilization are fully consistent with those 
 measure by a -arrestin recruitment assay , with concentration response 
 slopes that are commensurate with aggregation - based inhibition of 
 many other targets . of the four well - studied aggregators used in 
 this study , the flavonoid 
 quercetin is among the most promiscuous , appearing in hundreds of 
 papers with well over 50 reported targets ; among these , it has been reported to act against at least 12 different 
 gpcrs , including glp-1 receptor , 5ht2a serotonin receptor , 
 2a adrenergic receptor , and the d2 dopamine 
 receptor . whereas quercetin is a well - behaved kinase inhibitor at mid - nanomolar 
 concentrations , at micromolar concentrations its broad promiscuity knowing that quercetin acted via a colloid - based mechanism against 
 ccr4 , cx3cr1 , and v2r , we wondered whether related flavonoids 
 that have been reported to act on gpcrs would themselves form colloids . we therefore tested four flavonoids reported to be active on gpcrs 
 for colloid formation . at low to midmicromolar concentrations , two 
 of these , genistein and luteolin , formed particles by dls and inhibited 
 two counter - screening enzymes , -lactamase and cruzain , in the 
 same midmicromolar range , as they are observed to be active against 
 multiple gpcrs ( table 3 ) . also consistent with colloid formation , enzyme 
 inhibition was eliminated by the addition of 0.01% ( v / v ) trition x-100 
 and 0.025% ( v / v ) tween-80.table 2colloids sequester 
 peptidic ligandsasampleradius ( nm)% -lactamase 
 activityvasopressin0.18    0.1101.7    3.7tipt56.1    3.123.0    12.1tipt + vasopressin97.3    9.493.0    2.8athe particle 
 radii measured by 
 dls and the corresponding effects on -lactamase activity are 
 listed for vasopressin and tipt alone and in combination . the particle 
 radii measured by 
 dls and the corresponding effects on -lactamase activity are 
 listed for vasopressin and tipt alone and in combination . the colloid 
 radii measured by 
 dls and the effects on enzyme activity in the presence and absence 
 of detergent are shown . colloidal inhibition , certainly 
 of soluble proteins , arises from the sequestration of the protein 
 target itself , in a partly denatured form , by the colloidal particle . since ccr4 , cx3cr1 , and v2r are all peptidic gpcrs , we 
 wondered if their antagonism arose from association of the colloids 
 with the receptors themselves or with their peptide agonists . to test 
 for an interaction between colloid and peptide , we used dls to measure 
 the size of tipt colloids alone and mixed with vasopressin . the colloids 
 formed by tipt substantially increase in size when mixed with vasopressin , 
 from 56.1 to 97.3 nm in radius , suggesting that the peptide monomers 
 are being sequestered onto the colloidal surfaces ( table 2 ) . we also measured the inhibition of -lactamase 
 by tipt colloids with and without preincubation with vasopressin ; 
 proteins  and perhaps peptides  can prophylactically coat 
 colloids , preventing inhibition of a second enzyme . tipt at 10 m , 
 above the cac where colloids appear , leads to 77% inhibition of -lactamase ; 
 preincubating the colloidal mixture with vasopressin almost fully 
 blocks this inhibition ( 7% inhibition ) , suggesting that the peptide 
 binds the colloidal surface and competes with -lactamase binding . these observations support the idea that the colloids can directly 
 associate with the peptide ligands , which could contribute to inhibition 
 by sequestering and depleting agonist . while peptide sequestration 
 can thus contribute to apparent gpcr 
 antagonism , the colloids may also associate with the receptors themselves , 
 as they do with soluble enzymes . to investigate this , we measured 
 the effect of the four aggregators on basal gpcr signaling , which 
 occurs in the absence of agonist . three of the four aggregators , clotrimazole , 
 itraconazole , and tipt , inhibited basal receptor activity , acting 
 as apparent inverse agonists , with ic50 values of 1.6  
 3.2 , 0.2  2.0 , and 1.6  6.3 m , respectively ( figure 5 ) . these observations support a mechanism where 
 the colloids directly associate with the gpcrs themselves , acting 
 as inverse agonists . this mechanism is sufficient to explain their 
 inhibitory activity , though peptide - agonist sequestration may also 
 play a role . response curves for ccr4 in the -arrestin 
 recruitment assay in the absence of the ccr4 agonist , 
 ccl22 ( sem ) . the depression of basal activity by the colloid - formers 
 in the absence of the agonist suggests inverse agonism . the point of this study 
 is uncomplicated : colloidal aggregation , 
 well - appreciated as a major artifact in ligand discovery against soluble 
 proteins , can also affect gpcrs . first , well - studied colloid - formers inhibit a peptide- 
 and two protein - activated gpcrs in a colloid - dependent manner : inhibition 
 only occurs in the concentration range where these molecules aggregate 
 and , in well - controlled -arrestin recruitment assays , inhibition 
 is disrupted by removing the colloidal particles from solution , either 
 by centrifugation or by addition of detergent . in a second , complementary 
 line of investigation , flavonoids reported as active against other 
 gpcrs are shown to form colloids directly , by dynamic light scattering 
 ( dls ) and by detergent - dependent inhibition of classic counter - screen 
 enzymes like -lactamase . taken together , these results suggest 
 that colloid - based activity may contribute to artifactual ligand discovery 
 against g protein - coupled receptors , which remain crucial targets 
 for probe and drug discovery . if the key point of this paper 
 is simple , it nevertheless took 
 substantial effort to learn how to perturb the assays in such a way 
 as to demonstrate a colloid - based mechanism . whereas it is straightforward 
 to show activity of a colloid in a gpcr assay , mechanistically attributing 
 this to colloids demanded techniques to disrupt them or remove them 
 from solution . the twin techniques of tween-80 addition and of centrifugation  which 
 may be accomplished on a benchtop microcentrifuge  were key 
 to enabling this study , and are brought together here for the first 
 time . both have the virtue of simplicity , and may be deployed on a 
 large scale . several caveats merit mentioning . 
 whereas the mechanism of colloidal 
 inhibition of soluble proteins is relatively well - understood sequestration 
 of the protein by the colloid in a partly denatured form  their mechanism of action 
 on gpcrs is only partly illuminated by this study . the observation 
 that they act as inverse agonists , in the absence of activating agonist , 
 supports a direct role on the gpcrs themselves . however , unless the 
 colloids actually strip the gpcrs from the membrane , this can not be 
 by exactly the same mechanism as for sequestration of soluble proteins . indeed , activity via membrane perturbation can not be discounted , though 
 in control experiments colloids showed no gross effect on cell morphology 
 or viability . nor can activity via the agonist itself be discounted  indeed , 
 our results suggest that at least peptide agonists will be sequestered 
 by the colloids , possibly contributing to inhibition . in some assay 
 formats , even here , 
 in the calcium - mobilization assay , though the aggregating compounds 
 were just as effective as in the -arrestin recruitment assay , 
 reversibility was harder to see . we suspect this owes to the presence 
 of dyes in the kit used , but this is hard to deconvolute owing to 
 the proprietary nature of this kit . more broadly , cell - based assays 
 that include a large amount of protein , such as are found in serum - related 
 media , will be less susceptible to colloidal - aggregation ; though the 
 colloids themselves often persist in these and related media , their activity is often reduced , 
 if not fully eliminated . the assays used 
 in this study , however , remove the serum - supplemented media and replace 
 it with serum - free media before treatment with compound and therefore 
 remain quite sensitive to aggregation - based effects . finally , it is 
 uncertain what range of gpcrs will be affected by colloidal aggregation ligand 
 gpcrs , though the activity of promiscuous flavonoids on other families 
 of gpcrs suggests that their effects might be more broad . these 
 caveats should not obscure the key observation of this study : 
 colloidal aggregation can lead to artifactual activity against gpcrs . given the prevalence of aggregators in screening libraries , the artifactual activity of colloidal aggregators against gpcrs 
 could not only waste much effort and time , but may mask the activities 
 of weaker but better behaved compounds in screening campaigns . more 
 positively , the detergent and spin - down techniques described here 
 may be readily deployed on a large scale and can rapidly eliminate 
 most artifacts acting by this mechanism . given the importance of these 
 targets in biology and medicine , this mechanism of action against 
 gpcrs merits close attention . clotrimazole was purchased from mp biomolecules ; itraconazole was 
 purchased from ak scientific ; arg - vasopressin , quercetin , and tipt 
 were purchased from sigma . htla cells were cultured in dmem , supplemented 
 with 10% fetal bovine serum ( fbs ) , 100 units / ml penicillin , 100 mg / ml 
 streptomycin , 5 mg / l puromycin , and 100 mg / l hygromycin ( htla media ) . recruitment of -arrestin 
 via agonist - stimulated receptors ( ccr4 , cx3cr1 , and v2r ) 
 was quantified using the previously described -arrestin recruitment 
 assay . briefly , htla cells stably expressing 
 a -arrestin2-tev protease fusion protein and a tta - dependent 
 firefly luciferase reporter gene were plated in htla medium . using 
 a capo4 method reported earlier , cells were transfected with 18 g of the receptor - v2-tta construct and incubated overnight at 37 c , 5% co2 ( day 1 ) . the following day , cells were plated ( 20  000 cells / well , 
 50 l / well ) in white , clear - bottom , plk - coated , 384-well plates 
 in dmem containing 1% dialyzed fbs and antibiotics and incubated overnight 
 at 37 c , 5% co2 ( day 2 ) . on day 3 , the media was 
 replaced by serum - free dmem containing antibiotics , and cells were 
 allowed to recuperate for 23 h. serial dilutions of the reference 
 and test ligands ( 0.001 nm to 100 m , final concentrations , 
 prepared as 6 ) were made in sterile drug buffer ( 1 hbss , 
 20 mm hepes , 6% dmso , ph 7.4 ) . for the centrifugation treatment , concentrated 
 solutions of the ligands were spun at 13  000 g for 20 min on a benchtop microfuge at room temperature . for the 
 detergent treatment , tween-80 was added to concentrated solutions 
 of the ligands for a final concentration of 0.025% ( v / v ) . cells were 
 stimulated with 10 l / well of reference or test ligand , treated 
 or untreated , and incubated for 20 min . cells were stimulated a second 
 time with reference ligand at 1 m and incubated overnight at 
 37 c , 5% co2 . on day 4 , the media was removed from 
 the wells and 20 l of 10 brite - glo reagent ( promega ) 
 was added . cells were incubated for 30 min at rt and luminescence 
 was read using a trilux plate - reader ( 1 s / well ) . data were normalized 
 to vehicle ( 0% ) and reference ligands ( 100% ) and regressed using the 
 sigmoidal dose response function built into graphpad prism 5.0 . to measure 
 the effect of tween-80 
 on colloid formation , compounds were diluted from concentrated stocks 
 in dmso into filtered 1 hbss containing 20 mm hepes , ph 7.4 , 
 with and without 0.025% ( v / v ) tween-80 . the final dmso concentration 
 was 1% , and compound concentrations were as follows : 30 m tipt 
 and clotrimazole , 1 m itraconazole , 100 m quercetin . tipt , genistein , and 
 luteolin samples were measured in filtered 50 mm potassium phosphate , 
 ph 7.0 . to measure the effect of vasopressin on the size of tipt colloids , 
 light scattering was measured for 10 m tipt with and without 
 1 m vasopressin ; 1 m vasopressin was measured alone 
 in phosphate buffer as well . all measurements were made at room temperature using a dynapro ms / x 
 ( wyatt technology ) with a 55 mw laser at 826.6 nm . the laser power 
 was 100% , and the detector angle was 90. ampc -lactamase inhibition 
 was measured in 50 mm potassium phosphate , ph 7.0 , at room temperature . tipt ( 10 m , final concentration ) was diluted from a 10 mm stock 
 in dmso . -lactamase was measured with and without a five minute 
 preincubation with 1 m vasopressin . -lactamase ( 1 nm ) 
 was added to the samples and the mixture was incubated for 5 min ; 
 the reaction was initiated by adding 69 m centa substrate ( tydock 
 pharma ; modena , italy ) . genistein and luteolin were diluted from concentration 
 dmso stocks to obtain full dose  response curves against -lactamase 
 with final concentrations of dmso of 1% ( v / v ) . compound was incubated 
 with 0.2 nm -lactamase for 5 min , with and without 0.01% ( v / v ) 
 triton x-100 , and the reaction was initiated by adding 46 m 
 centa . the final reaction volumes were 1 ml . change in absorbance 
 was monitored at 405 nm for 2.5 min using an hp 8453 uv cruzain inhibition 
 was measured in 0.1 m sodium acetate , ph 5.5 , containing 5 mm dtt . genistein and luteolin were incubated with 0.8 nm cruzain ( with and 
 without 0.025% ( v / v ) tween-80 ) for 5 min until reactions were initiated 
 by adding the fluorogenic substrate z - phe - arg - aminomethylcoumarin 
 ( z - fr - amc ) . the final reaction volume was 200 l . final concentrations 
 were 0.4 nm cruzain , 2.5 m z - fr - amc , and 1% dmso . enzyme inhibition 
 was measured by monitoring the increase in fluorescence for 5 min 
 ( excitation wavelength of 355 nm , emission wavelength of 460 nm ) in 
 a microtiter plate spectrofluorimeter ( flexstation , molecular devices ) . assays were performed in duplicate in 96-well plates and were repeated 
 in two separate experiments . response curves were plotted , 
 and ic50 values were calculated using graphpad 
 prism 4 ( graphpad , san diego , ca ) using a sigmoidal dose  response 
 curve analysis with variable slope .
colloidal 
 aggregation is the dominant mechanism for artifactual 
 inhibition of soluble proteins , and controls against it are now widely 
 deployed . 
 conversely , investigating this mechanism for membrane - bound 
 receptors has proven difficult . here 
 we investigate the activity of 
 four well - characterized aggregators against three g protein - coupled 
 receptors ( gpcrs ) recognizing peptide and protein ligands . 
 each of 
 the aggregators was active at micromolar concentrations against the 
 three gpcrs in cell - based assays . 
 this activity could be attenuated 
 by either centrifugation of the inhibitor stock solution or by addition 
 of tween-80 detergent . in the absence of agonist , 
 the aggregators 
 acted as inverse agonists , consistent with a direct receptor interaction . 
 meanwhile , several literature gpcr ligands that resemble aggregators 
 themselves formed colloids , by both physical and enzymological tests . 
 these observations suggest that some gpcrs may be artifactually antagonized 
 by colloidal aggregates , an effect that merits the attention of investigators 
 in this field .