g protein - coupled receptors ( gpcrs ) form 
 a large and important protein family with 390 members ( excluding olfactory 
 receptors ) in the human genome .
gpcrs 
 are critically involved in cell signaling in response to a wide range 
 of endogenous ligands , including hormones , neurotransmitters , cytokines , 
 odorants , and light .
they fall into three major classes ( families 
 a , b , and c , of which family a , the rhodopsin family , is the largest ) . 

historically , the gpcr arena has been a fruitful one for drug discovery , 
 with a large number of small molecule drugs being successfully developed 
 in multiple therapeutic areas , together with a smaller number of new 
 biological entities .
gpcr drug discovery continues to be an area of 
 significant focus for the pharmaceutical industry , with over 60 new 
 gpcr drugs , almost a quarter of the total number of approved new chemical 
 entities ( nces ) in this period , being launched in the past 10 years . despite this success ,
only one new gpcr target 
 per year on average has been drugged in the past decade , indicating 
 that gpcr research remains a difficult area and one in which there 
 are many targets with clinical relevance and validation that are currently 
 underserved by drug discovery efforts .
historically , the vast majority 
 of gpcr drug discovery efforts have relied upon cell - based assays 
 coupled with high - throughput screening of large compound libraries 
 for hit identification .
this approach has resulted in limited success 
 for challenging , clinically valuable targets such as neuropeptide 
 receptors , chemokine receptors , family b peptide - hormone receptors 
 and family c metabotropic glutamate ( mglu ) receptors . until 
 recent years , in contrast to soluble protein classes such as enzymes , 
 x - ray crystal structures of gpcrs had been lacking with only the structure 
 of the visual pigment rhodopsin , first reported in 2000 , being available 
 to guide structure - based drug discovery efforts . after a gap of 7 years , structures of the 2-adrenergic receptor ( 2ar ) were published , rapidly followed by the revelation of medium to high resolution 
 crystal structures of 14 additional receptors ( 1-adrenergic ( 1ar ) , adenosine a2a , sphingosine 1-phosphate 1 ( s1p1 ) , chemokine cxcr4 , dopamine 
 d3 , histamine h1 , muscarinic acetylcholine m2 and m3 , neurotensin , opioid receptors  ,  ,  , nociceptin , and protease - activated receptor 1 ( par1 ) .
it has also recently been disclosed that the 
 first example of a family b gpcr structure , that of the corticotropin 
 releasing factor ( crf-1 ) receptor , has been solved .
the significance of this recent upsurge in gpcr crystallographic 
 information to drug discovery has been recently reviewed .
the success in obtaining new x - ray crystal structures has 
 been due to technical advances that have stabilized purified receptors 
 in detergent solution and locked them in specific conformations , both 
 of which are necessary for the formation of well - diffracting crystals . two key approaches to enable the determination of gpcr structures 
 have been devised in recent years .
first , insertion of the enzyme 
 t4 lysozyme ( t4l ) into intracellular loop 3 ( icl3 ) of the receptor 
 to promote the formation of crystal contacts has been used . the high 
 resolution ( 2.4  )
structure of the 2ar , previously solved at 3.4  in complex with 
 an antibody fragment , was the first demonstration 
 of this strategy .
the t4l approach has been used in combination with 
 the lipidic cubic phase ( lcp ) crystallization method .
the second approach 
 introduces a small number of point mutations into the receptor construct , 
 significantly increasing thermostability of the protein and thereby 
 enabling purification in the short chain detergents required for crystallization 
 by vapor diffusion .
thermostabilized receptors form well - ordered crystals , 
 as first demonstrated in the structure determination of the turkey 
 1ar at 2.7  resolution .
the thermostabilization approach has the advantage of locking 
 the receptor into a single homogeneous conformation , determined by 
 the pharmacology of the ligand used in the stabilization process , 
 greatly facilitating purification and structure determination
. stabilized 
 receptors formed via this approach are also known as stars and typically contain a small number of thermostabilizing 
 mutations , six in the case of the 1ar star ( 1ar - m23 ) , which is stabilized in an antagonist conformation .
adrenergic receptors ( adrenoceptors ) are 
 family a gpcrs that activate intracellular g proteins upon binding 
 of endogenous catecholamine ligands such as adrenaline and noradrenaline .
the adrenergic receptors are split into  
 and  classes , with the latter having 1 , 2 , and 3 subtypes .
more than 4 decades of 
 research and development in the area of -adrenergic receptors 
 have resulted in a legacy of clinically important agonist and antagonist 
 molecules with varying degrees of selectivity .
antagonists of -adrenergic receptors are frequently 
 used in cardiovascular medicine , as well 
 as in other areas such as migraine and anxiety , and -agonists 
 are used in the treatment of asthma and 
 chronic obstructive pulmonary disease ( copd ) .
crystal structures of the human 2ar and turkey 1ar in 
 complex with the inverse agonist carazolol 1 and antagonist 
 cyanopindolol 2 , respectively ( chart 1 ) , were reported in 2007 and 2008 .
subsequently , structures 
 with the full agonists carmoterol 3 and isoprenaline 4 and the partial agonists salbutamol 5 and dobutamine 6 in the 1ar were published ( chart 1 ) , as were structures with 
 carazolol and the antagonist iodocyanopindolol in the same receptor .
2ar crystal structures with 
 the inverse agonist ici118,551 and agonist bi-167107 , the latter in 
 a nanobody - stabilized active state of the 2ar , have 
 been published .
more recently , structures of 
 the stabilized 1ar bound to the  blockers 
 bucindolol and carvedilol , which are classified as biased agonists 
 because they stimulate g protein - independent signaling , have been 
 reported . in comparison to other 1ar structures ,
both of these ligands make additional contacts 
 to helix 7 and extracellular loop 2 ( el2 ) of the receptor , an observation 
 that may provide an insight into the structural requirements of biased 
 ligands . taken together , the 1ar and 2ar structures provide a wealth of structural understanding 
 within the -adrenergic area , including insights into ligand 
receptor 
 interactions that impart antagonist , partial agonist , or full agonist 
 pharmacologies .
the advent of gpcr crystal structures has initiated a new 
 era of structure based drug design ( sbdd ) for this important target 
 class . in concert with sbdd , fragment - based drug discovery ( fbdd ) 
 as a strategy for identifying small ( 100250 da ) , efficient 
 hit molecules is now a well established technique .
fragment hits , when coupled with a structural understanding of how 
 they interact with their target protein , represent excellent starting 
 points for medicinal chemistry , and many examples of how fragments 
 have been successfully optimized to potent leads have been published .
indeed , multiple agents have been progressed into clinical trials 
 in recent years and the first fragment - derived drug , vemurafenib , 
 has reached the market .
biophysical methods 
 such as spr , nmr , and x - ray crystallography are among the mainstays 
 of fbdd approaches used for soluble protein targets , but their application 
 to the gpcr field has been highly challenging because of low expression 
 and poor stability of the target receptor when isolated from the cell 
 membrane . for these reasons , fbdd has been rarely utilized for gpcrs 
 to date .
the engineering 
 of stars with significantly increased thermostability has recently 
 enabled fragment screening of gpcrs to be validated for the first 
 time using a variety of biophysical and biochemical approaches . screening 
 of the 1ar and adenosine a2a receptors 
 by target - immobilized nmr screening ( tins ) and spr techniques , respectively , 

was reported in 2011 , and fragment screening 
 of the adenosine a2a receptor has been recently validated 
 using the capillary electrophoresis ( ce ) approach .
additionally , capture of the 1ar 
 star and evaluation of the binding constants of small - molecule antagonists 
 by spr have been described , and by use 
 of this approach , a subset of the heptares fragment library ( approximately 
 650 fragments ) was screened in tandem against the 1ar star and adenosine a2a receptor star .
figure 1 shows the responses obtained from the 1ar surface plotted against responses from the a2a receptor surface .
most responses ( some of those shown in gray squares ) 
 cluster around the origin .
a number of responses ( also 
 shown in gray squares ) showed binding to both a2a and 1ar .
positive control compounds for each receptor were screened periodically 
 and gave responses that cluster near the axes , confirming that these 
 two compounds were selective binders to either 1ar or a2a receptor .
similarly , a subset of fragments appeared 
 to bind more significantly to one receptor or the other ; hits highlighted 
 in red were selective a2a binders , those highlighted in 
 yellow were selective 1ar binders .
the selective 
 1ar hits were retested in concentration series to 
 obtain affinity information . among the selective binders to the 1ar star were 1-[3-(trifluoromethyl)phenyl]piperazine and 2-(piperazin-1-yl)quinoline 
 ( 7 and 8 , chart 2 ) , which were estimated to have encouraging binding affinities ( kd = 16 m ( pkd = 4.80 ) and 5.6 m ( pkd = 5.25 ) , 
 respectively ) and good ligand efficiency ( le = 0.41 and le = 0.48 , 
 respectively ) .
plot of spr responses 
 of heptares fragment library against the adenosine a2a star 
 and 1ar star proteins : orange circles , 1ar selective hits ; blue squares , 1ar control 
 compound ; red circles , a2a selective hits ; green squares , 
 a2a control compound .
substituted phenylpiperidines have ample history within 
 gpcr drug discovery and have been described as privileged structures 
 for this target class .
the analogous 
 phenylpiperazines also have precedent within gpcr chemical space , 
 for example , in the atypical antipsychotics aripiprazole and lurasidone ( chart 2 ) . despite this significance ,
phenylpiperazines 
 have little precedent in the field of -adrenergic receptors , 
 where the vast majority of clinically relevant molecules are typified 
 by an ethanolamine side chain extending from an aromatic or heteroaromatic 
 moiety ( e.g. , chart 1 , 15 ) .
encouraged by the affinity and efficiency of spr hits 7 and 8 , we embarked upon a fragment hit - to - lead 
 exercise to explore these phenylpiperazine fragments as novel hits 
 for the 1ar .
screening in an orthogonal assay 
 format is a routine approach to confirm hits during fbdd , and in line 
 with this strategy we assembled a set of commercially available analogues 
 of similar size and complexity compared to 7 and 8 ( see modeling section ) .
the set 
 was screened in a radioligand membrane binding assay with human wild - type 
 1ar and [ h]dihydroalprenolol , and affinity 
 data ( together with ligand efficiency ( le ) , ligand lipophilicity efficiency ( lle ) , and clogp values ) for selected analogues 
 are shown in table 1 .
all of the commercially 
 available analogues tested have high ligand efficiencies and bind 
 with moderate to high affinity .
unsubstituted phenylpiperazine 9 has good affinity , with the more polar 2-pyridyl and 2-pyrimidinyl 
 analogues of 7 ( 10 and 11 , 
 respectively ) being weaker and less ligand efficient than 9 .
disubstituted phenyl groups yielded the highest affinities , with 
 chloro ( 1214 ) and methyl ( 15 , 16 ) variants yielding higher affinities than 
 the trifluoromethyl or methoxy derivatives 17 and 18 .
the chloro or methyl disubstituted analogues , where 2,3- 
 or 3,5-regiochemistry is preferred over the 3,4-isomers , bound with 
 moderate to high affinity and exceptional ligand efficiencies ( > 0.60 ) . 

ligand lipophilicity efficiency ( lle = pki  clogp ) is another measure of druglikeness ; in general lles for the fragments are moderate to good 
 ( with the exception of the highly lipophilic 17 ) .
the 
 le and lle values for several compounds in table 1 compare favorably to those of the well characterized 1ar ligands carazolol 1 ( le = 0.62 , lle = 6.4 ) , 
 cyanopindolol 2 ( le = 0.56 , lle = 6.8 ) , and carmoterol 3 ( le = 0.38 , lle = 5.6 ) .
selected quinoline derivatives to 
 explore initial spr hit 8 were screened ; 8 was included as a control and returned highly comparable affinity 
 in the radioligand binding assay to that from the spr screen ( pki = 5.20 vs pkd = 
 5.25 , respectively ) .
installation of a methyl group at the 4-position 
 ( 20 ) or substitution at the 8-position ( 21 ) yielded compounds with approximately 25-fold greater affinity than 8 , whereas interchanging the methyl and piperazinyl groups 
 of 20 to yield isomer 22 was only moderately 
 effective .
compounds 23 and 24 were purchased 
 to loosely mimic the carbazole core of carazolol ; closer analogues 
 were not readily available . nevertheless , 23 and 24 returned submicromolar affinity , albeit with lower lle 
 than the parent hit 8
. selected compounds ( 12 , 13 , 19 , and 20 ) were profiled 
 in muscarinic m1m4 acetylcholine receptor 
 membrane binding and agonist functional assays to provide an initial 
 indication of selectivity of the piperazine fragments against related 
 family a gpcrs .
all four compounds were inactive in the functional 
 assay for each isoform and had only weak affinity at best in the binding 
 assays ( 13 , 19 , and 20 m1m4 pki < 
 4.7 ; 12 m1m3 pki = 5.15.4 , m4 pki = 4.5 ) .
given the precedent for related piperazines 
 such as aripiprazole and lurasidone to have complex pharmacology with 
 activities against -adrenergic , dopaminergic , and serotonergic 
 receptors , characterization of optimized compounds in the series against 
 these gpcr subfamilies will be relevant .
the advent of gpcr crystallographic information presents opportunities 
 for structure - based design and discovery .
we sought to use the available 1ar crystal structure 
 data to drive progress with the piperazine fragment hits .
as it is 
 not currently feasible to perform crystallography on the human 1ar because of its instability , these data were obtained with 
 the turkey 1ar .
however , the two receptors are 82% 
 identical in the transmembrane and loop regions ( with the exclusion 
 of icl3 ) and the residues that comprise the ligand binding pocket 
 are 100% identical .
the turkey receptor therefore provides an excellent 
 model for studying ligand  receptor interactions .
receptor interactions observed 
 in the 1ar structures with cyanopindolol , carmoterol , 
 and carazolol bound were of notable interest . during the hit - to - lead 
 process
, molecules were routinely docked into the liganded structure 
 of the 1ar and examined 
 in relation to inverse agonist carazolol 1(8 ) and agonist carmoterol 3 . because of the small size of fragments and the 
 multitude of ways they can fit into a receptor binding site , it is 
 notoriously difficult to dock these ligands with any great confidence 
 without examining the site in question in significant detail .
a complete 
 druggability analysis of the binding 
 site was therefore carried out to identify the region most likely 
 to be occupied by the fragment .
water and ch aromatic probes show 
 the regions most favorably occupied by polar and hydrophobic portions 
 of a ligand , respectively .
optimization of overlap of the docked molecule 
 along with displacement of the least energetically favorable waters 
 ( shown in red in figure 2 ) has been shown previously 
 to be predictive for highly ligand efficient molecules .
thus , this strategy was also used in our analysis 
 of the solutions obtained from dockings , as described below in further 
 detail for representative hit 16 .
additional compounds 
 were selected from similarity and substructure searching approaches 
 to complement the structure - based strategy and expand sar in the series .
druggability 
 analysis of the 1ar ligand binding site , showing water molecules and their energies relative 
 to bulk solvent ( red , high ; blue , low ) combined with surfaces depicting 
 water probe ( green ) and ch aromatic probe ( yellow ) hotspots , with 
 the docking pose of 16 selected for comparison to the 
 crystal structures . in the dockings of 16 with carazolol 1 and carmoterol 3
the carbon atom of the 3-methyl substituent of 16 overlays 
 clearly with the carbazole nitrogen of 1 and is in the 
 region of the quinolinone nitrogen of 3 , suggesting an 
 opportunity to introduce similar polar interactions with the receptor 
 in this area of the molecule ( figure 3 ) .
this 
 possibility is more clearly visualized in the structure of cyanopindolol 2 in the 1ar stabilized receptor ( figure 4 ) , where hydrogen 
 bond donation of the indole nitrogen to ser211 ( superscripts 
 indicate ballesteros  weinstein nomenclature ) , a nonpolar interaction of the indole phenyl ring with 
 phe307 , and interaction of the charged ethanolamine 
 portion with asp121 and asn329 are observed . 

inspired by these observations , we extended our studies to include 
 a number of molecules with larger and more polar substituents in order 
 to promote further interactions with residues in the 1ar ligand binding site and in particular polar interactions with 
 ser211 on helix 5 .
the strategy most notably resulted in identification 
 of the simple indole derivative 19 ( table 1 ) , which has both high affinity and ligand efficiency and 
 reduced lipophilicity compared to parent molecule 16 , 
 a finding that validated our structure - driven approach and represents 
 a very promising lead compound for further optimization .
piperazine 16 ( pink carbons ) docked into the crystal structures of ( a ) 
 1ar complexed with the inverse agonist carazolol 1 ( orange carbons ) ( pdb code 2ycw ) and ( b ) 
 1ar complexed with the agonist carmoterol 3 ( cyan carbons ) ( pdb code 2y02 ) .
( a ) x - ray crystal structure of the antagonist cyanopindolol 2 in complex with 1ar ( pdb code 2vt4 ) .
the purple arrows 
 and their directions represent hydrogen bonds between the ligand and 
 the protein .
the green line represents the  
 stacking arrangement seen between the aromatic core and the aromatic 
 residue phe307 .
the identification of a series 
 of piperazine - based fragments with high affinity and high solubility provided an invaluable opportunity to obtain 
 co - structures of the molecules with the 1ar , as 
 detailed structural information would confirm the modes of binding 
 of the fragments , facilitate comparisons with existing structures , 
 and indicate opportunities for further development . on the basis of 
 a consideration of affinity and ligand efficiency , indole 19 and the structurally dissimilar quinoline 20
cocrystallization of thermostabilized 
 1ar with fragments 19 and 20 was performed as previously described and resulted 
 in structures determined to resolutions of 2.8 and 2.7  , respectively 
 ( supporting information table 2 ) .
the structures 
 show that the piperazine rings in the two molecules are located between 
 asp121 on helix 3 ( h3 ) and asn329 on 
 h7 , and their phenyl substituents are positioned adjacent to h5 ( figure 5a and figure 5b ) .
the piperazine 
 ring therefore substitutes for the ethanolamine core of conventional 
 adrenergic receptor ligands , and the structural roles of the phenyl 
 substituents are similar to those of the indolecarbonitrile and carbazole 
 headgroups of cyanopindolol and carazolol ( figure 5c and figure 5d ) .
however , the total 
 number and nature of ligand  receptor interactions observed 
 in the 1ar-19 and 1ar-20 structures are reduced when compared to those 
 observed in the crystal structures with cyanopindolol and carazolol ( supporting information table 3 ) .
with cyanopindolol 
 and carazolol , the ethanolamine secondary amine and -hydroxyl 
 groups both form hydrogen bonds to the asp121 and asn329 side chains .
in contrast , with 19 and 20 , only one secondary amine nitrogen in the piperazine ring 
 is suitably positioned to interact with asp121 and 
 asn329 , and the interactions are mostly weaker , polar 
 interactions rather than hydrogen bonds ( supporting 
 information table 3 ) .
possibly as a consequence of the weaker 
 interactions of fragments 19 and 20 , there 
 is greater variation in the orientation of the piperazine rings than 
 is shown by the ethanolamine groups in carazolol and cyanopindolol . 

this can be observed in the superpositions of the different ligands 
 and fragments complexed with the receptor depicted in figure 5e .
the variation in the positions of the piperazine 
 rings in the two structures results in some differences in how 19 and 20 interact with asp121 , asn329 , trp303 , and tyr333 ( supporting information table 3 ) .
the 
 structures of 1ar bound to 19 and 20 do not show either the change in rotamer conformation of 
 ser215 observed when full agonists are bound or a contraction of the ligand binding pocket 
 ( supporting information table 3 ) characteristic 
 of the binding of both full and partial agonists .
these observations suggest that both of the fragments are 
 antagonists , although data to support this hypothesis have not yet 
 been generated .
however , the interactions of the phenyl substituent 
 headgroup with ser211 on h5 differ between the two 
 fragment molecules , as only 19 can form a hydrogen bond 
 to ser211 . in the 1ar-20 structure ,
the conformation of ser211 is similar 
 to that observed in the 1ar - carazolol structure 
 ( figure 5b and figure 5d ) , whereas in the 1ar-19 structure 
 the conformation of ser211 corresponds to that observed 
 in the 1ar - cyanopindolol structure ( figure 5a and figure 5c ) .
the differences 
 in pharmacological activity between cyanopindolol ( a partial agonist 
 with sympathomimetic activity ) and carazolol ( an inverse agonist ) 
 may in part result from the differences in the conformation of ser211 observed in the structures with these ligands ( figure 5c and figure 5d ) , and there may be similar differences in pharmacological 
 activity between 19 and 20 .
because of their 
 relatively small size , fragments 19 and 20 interact with a subset of the residues that comprise the ligand 
 binding pocket .
this suggests that by use of the detailed structural 
 information that has been made available , the targeted addition of 
 further substituents to these fragments could serve to further increase 
 their affinities , as well as to modulate their pharmacological activities .
1ar structures are shown in cartoon representation as viewed 
 in the membrane plane with all or part of h1 , h2 , h3 , and h4 obscuring 
 the ligand binding site removed for clarity , and the extracellular 
 side is uppermost : ( a ) 1ar-19 ; ( b ) 
 1ar-20 ; ( c ) 1ar - cyanopindolol 
 ( pdb code 2vt4 ) ; ( d ) 1ar - carazolol 
 ( pdb code 2ycw ) .
atoms are colored accordingly . for 
 ligands / fragments : c , cyan , gold , silver , yellow ; o , red ; n , blue . 
 for selected receptor side chains : c , green ; o , red ; n , blue .
potential 
 hydrogen bonds and polar contacts are shown as red dashes . for a full 
 list of receptor  ligand interactions see supporting information table 3 .
. 
 global alignment of the receptor structures ( monomers selected as 
 in supporting information table 3 ) was 
 performed using pymol ( superpose ) , and the resulting positions of 
 the ligands are depicted .
fragment screening is a well - established 
 and powerful approach to the discovery of a new lead series that is 
 now starting to be utilized for membrane - bound receptor targets .
recent 
 virtual screening efforts have demonstrated that , at least for aminergic 
 family a gpcrs , fragment hits can be identified , now that the details 
 of how small molecules bind to these receptors are better understood .
furthermore , studies on , for example , the histamine h1 receptor ( h1r ) have shown , in a manner analogous to the 
 work exemplified here , that fragment - sized ligands can be bound to 
 the receptor , crystallized and their binding modes established . in the research presented here , biophysical 
 fragment screening of 1ar using spr was enabled 
 by receptor stabilization and led to the identification of arylpiperazine 
 hits 7 and 8 .
selection of analogues for 
 screening in an orthogonal wild - type radioligand membrane binding 
 assay through similarity and substructural searching approaches was 
 complemented by the use of -adrenergic receptor crystallographic 
 information , allowing a parallel structure - based design strategy . 

high affinity and ligand efficient fragments were identified , including 
 indole 19 and quinoline 20 , which were identified 
 and subsequently cocrystallized with the stabilized 1ar , yielding structures at 2.8 and 2.7  , respectively .
overall , 
 these results demonstrate , for the first time , that a true fragment 
 based drug discovery paradigm , encompassing biophysical screening 
 with a direct - binding platform of a diverse fragment library , structure - guided 
 fragment optimization , and cocrystallization of fragment hits , can 
 now be applied to gpcr targets .
hek293 cells were transiently transfected with cdna encoding human 
 1ar using genejuice ( novagen ) according to the manufacturer s 
 instructions to achieve approximately 2  10 post - transfected 
 cells . after 48 h of incubation
the cells were dissociated using tryple 
 express cell dissociation fluid and washed with pbs , pelleted down , 
 and stored at 80 c until required .
the pellet was resuspended 
 in chilled buffer consisting of 20 mm hepes , 10 mm edta containing 
 1 complete protease inhibitor cocktail tablet per 50 ml of buffer 
 ( ph 7.4 ) .
the suspension was homogenized for 15 s and centrifuged at 40000 g for 15 min .
the pellet was suspended in buffer containing 
 20 mm hepes and 0.1 mm edta ( ph 7.4 ) and homogenized for 30 s. after 
 centrifugation at 40000 g for 45 min , the pellet was 
 resuspended in the same buffer and homogenized for a further 30 s. 
 protein concentration was estimated against the standard bovine serum 
 albumin using the bca method ( pierce , socochim , lausanne , switzerland ) 
 and then frozen to 80 c at 2 mg / ml prior to use .
cell membranes were 
 incubated with [ h]dihydroalprenolol in assay buffer ( 50 
 mm hepes , 15 mm mgcl2 , 150 mm nacl , ph 7.4 ) in a total 
 assay volume of 0.25 ml with a final dmso concentration of 1% .
after 
 90 min of incubation at room temperature the reaction was terminated 
 by rapid filtration through gf / b 96-well glass fiber plates with 5 
  0.25 ml washes with doubly distilled h2o using a 
 tomtec cell harvester .
bound radioactivity was determined through 
 liquid scintillation using lablogic safescint and detected on a microbeta 
 liquid scintillation counter .
nonspecific binding was determined as 
 that remaining in the presence of a 10 m saturating concentration 
 of the antagonist alprenolol .
competition binding was performed by 
 incubating membranes ( 1.25 g of protein / well ) with 1.5 nm [ h]dihydroalprenolol and a range of concentrations of the test 
 compound .
ic50 values were derived from fitting to a four - parameter 
 logistic equation in prism ( graphpad software , san diego , ca , u.s . ) . 

binding 
 affinities are expressed as pki values , 
 where pki = log10  ki .
surface 
 plasmon resonance ( spr ) binding studies were performed at 25 c 
 using biacore t200 and s51 optical biosensors equipped with nickel - charged 
 nta chips and equilibrated with running buffer ( 20 mm tris - hcl , 350 
 mm nacl , 0.1% dodecylmaltoside , 5% dmso , ph 7.8 ) .
aliquots of the 
 affinity - purified , his - tagged 1ar and a2a receptors were both capture - coupled on the sensor surface to densities of > 9000 resonance units ( ru ) . 
 in an initial screen ,
the entire library was tested at 50 m 
 with two control compounds , l-748,337 ( a 1ar positive 
 control , cas 244192 - 94 - 7 ) and psb 1115 ( an a2a receptor 
 positive control , cas 409344 - 71 - 4 ) , tested periodically at 10 m 
 throughout the screen to track the activity of the receptor surfaces . 
 for a second screen ,
fresh receptor surfaces were prepared and the 
 compound library was retested at 11 m ( after omitting compounds 
 that bound to the reference surface or bound with nanomolar affinity 
 to a2a ) .
potential selective 1ar hits 
 were analyzed in concentration series ( 2-fold dilutions typically 
 starting at 150 m , with each concentration tested in duplicate ) 
 to confirm their selectivity and determine their affinities .
the crystal structure of 1ar 
 bound with cyanopindolol , pdb code 2vt4 , was used 
 as the basis for dockings of fragments .
the protein preparation workflow was used to 
 add hydrogens , cap the n - terminus and c - terminus groups , optimize 
 the orientation of hydroxyl groups , asn , and gln , and the protonation 
 state of his , and then perform a number of constrained refinements 
 on the hydrogens alone and the protein with a maximum rmsd tolerance 
 of 0.30  .
dockings were undertaken using the glide extra precision 
 ( xp ) protocol within maestro .
grid analyses of the binding sites were 
 used to evaluate potential docking poses and drive the designs ( using 
 the csp3 probe ( c3 ) for shape , aromatic ch probe ( c1= ) for 
 lipophilic hotspots , water probe ( oh2 ) for water hotspots , 
 carbonyl group probe ( o ) for hydrogen bond acceptor hotspots , and 
 amide nh probe ( n1 ) for hydrogen - bond donor hotspots ) .
the turkey ( meleagris gallopavo ) 1ar ( star ) construct 
 that was used in crystallization experiments , 44-m23 , contains 
 six thermostabilizing point mutations and truncations at the n terminus , 
 inner loop 3 , and c terminus .
for crystallization , the detergent was exchanged to hega-10 ( 0.35% ) 
 on an alprenolol affinity column .
receptor was competitively eluted 
 from the alprenolol affinity column with 100 m ligand ( 19 or 20 ) and concentrated to 25 mg / ml in 10 
 mm tris - hcl , ph 7.6 , 100 mm nacl , 0.1 mm edta , 0.35% hega-10 . before 
 crystallization , hega-10 and cholesteryl hemisuccinate
crystals were grown at 4 c 
 by vapor diffusion in sitting drops with 150 nl of receptor + 150 
 nl of precipitant ( 0.1 m bicine , ph 9.0 , 24% peg 600 in both cases ) 
 and cryoprotected by addition of 60% peg 600 for 1 min before mounting 
 on hampton crystalcap ht loops and cryocooling in liquid nitrogen . for 
 both 1ar-19 and 1ar-20 structures ,
diffraction data were collected from 
 a single cryocooled crystal ( 100 k ) using a 10 m focused beam 
 at i24 , diamond light source , oxford , u.k .
both structures were solved by molecular replacement with 
 phaser using the structure of 1ar with carvedilol bound ( pdb code 4amj(39 ) ) as a starting 
 model .
refinement , rebuilding , and validation were carried out with 
 refmac5 , coot , and molprobity . in both the 1ar-19 and 1ar-20 structures
there is a distortion of the ligand binding pocket in 
 monomer a due to lattice contacts , and monomer b represents the more 
 physiologically relevant conformation .
the 1ar-19 and 1ar-20 crystal structures 
 were determined at a resolution of 2.8 and 2.7  , respectively . 

this resolution was more than adequate to provide clear omit electron 
 densities that enabled unequivocal placement of the ligands in the 
 structures ( supporting information figure 1 ) , as well as further details such as specifically bound detergent 
 and lipid molecules and ordered internal water molecules and sodium 
 ions ( supporting information table 2 ) .
 biophysical fragment screening of 
 a thermostabilized 1-adrenergic receptor ( 1ar ) using surface plasmon resonance ( spr ) enabled the identification 
 of moderate affinity , high ligand efficiency ( le ) arylpiperazine hits 7 and 8 . 
 subsequent hit to lead follow - up confirmed 
 the activity of the chemotype , and a structure - based design approach 
 using protein  
 ligand crystal structures of the 1ar resulted in the identification of several fragments that 
 bound with higher affinity , including indole 19 and quinoline 20 . in the first example of gpcr crystallography with ligands 
 derived from fragment screening , 
 structures of the stabilized 1ar complexed with 19 and 20 were 
 determined at resolutions of 2.8 and 2.7  , respectively . 
Introduction
Results and Discussion
Conclusions
Experimental
Section