in the post - genomic 
 era , emphasis is placed on understanding how 
 various biological macromolecules specifically interact and assemble , 
 either through protein  protein or protein  ligand associations , 
 to orchestrate dynamic responses that influence physiologic decision - making . this impetus has sparked new methods to interrogate how these seemingly 
 minor alterations drive phenotypic responses . the challenges in this field are manifold : there 
 are over 20  000 protein - coding genes in humans  and even more macromolecule or small - molecule potential 
 binding partners . all these components can modulate the functions 
 and dynamics of the interactome specific to individual gene products . thus , identifying target(s ) , binding sites , and the nature of the 
 input signal , and ultimately forging a precise link between these 
 myriad upstream events and a phenotypic output is no easy task . arguably , 
 the go - to chemical biology method to engender specificity in these 
 highly interconnected networks this perspective conceptualizes 
 the latest proximity - enhanced design strategies with an eye toward 
 stimulating further innovation and highlighting areas where improvements 
 are required . we split chemical tethering into two classes ( figure 1 ) and systematically 
 discuss the merits and drawbacks of both . through this organization , 
 we hope to help the researcher decide the ideal strategy to solve 
 a specific biological problem , and to further seed new inspirations 
 and spur on next - generation chemical biology innovations . in this perspective , 
 the chemical biology toolsets built upon proximity 
 enhancement are classified into two general categories , class i and 
 class ii . class i is further broken into two subclasses : in subclass 
 ia , the bifunctional probe enables recruitment of two or more distinct 
 biological entities ( proteins / cells ) , thereby promoting a response ; 
 and in subclass ib , one pole of the small molecule serves as an anchor 
 to the poi and the other pole reacts intramolecularly with the same 
 poi . in class ii , on - demand precision targeting enables the reactive 
 entity to be unmasked in situ and targeted to ( or 
 the microenvironment of ) poi . in all cases , in the absence of ligands 
 known to bind poi , ligands generic to various protein- and peptide - based 
 tags ( halo , clip , snap , prime , etc . ) that can be fused to pois may 
 be integrated into the probes . tethering is the blueprint of established proximity - augmented 
 heterofunctional 
 small - molecule probes . we define class i proximity enhancement to 
 be a system in which the chemical linker between the two poles of 
 a bifunctional small - molecule probe is unbroken throughout an experiment 
 ( figure 1 ) . there is 
 widespread use and diverse applications of the class i strategy in 
 biology , yet due to the rapid expansion of the field , it is timely 
 to re - stress the parallels of all these approaches at the chemical 
 level while tipping our hat to the huge diversity they can engender 
 at the biological level . the second act of this perspective broaches class ii proximity enhancement . 
 instead of employing permanently tethered small - molecule scaffolds , 
 class ii uses a bifunctional small molecule that contains a recognition 
 unit at one pole but at the other a latent warhead ( such as a sensitizer 
 or a photocaged precursor ) that can be activated in situ ( figure 1 ) . early 
 incarnations of this new concept have interrogated specific redox 
 response by targeted redox signaling on demand . related small - molecule - directed in situ unmasking ideas of reactive entities have also been 
 powerfully applied in characterizing nascent interactomes such as 
 mitochondrial proteome mapping and ribosome profiling . we focus on the common concepts that drive and ultimately limit 
 these emerging strategies . astoundingly , because the core chemical 
 concepts are similar , the respective classes show similar strengths 
 and limitations , implying that there may be cross - cutting solutions 
 to some of these problems . in most cases , the seminal proof of concept for the power of tethered 
 small - molecule 
 probes in biology was  chemical inducers of protein dimerization  
 ( cid ) . cid uses a bifunctional small molecule that 
 interacts with two separate proteins of interest ( pois ) to induce 
 the formation of an active complex or localization - specific recruited 
 state . the cid - induced protein dimerization event was shown to be 
 sufficient to prompt tripping of signaling nodes , eliciting selective 
 pathway activation . cid was first introduced in 1993 and proved to be a game - changer in the field that spawned 
 many tethered small - molecule modulators . these bifunctional tethered 
 probes were successfully put to task to help understand diverse biological 
 quandaries . herein we subcategorize the established scaffolding systems into 
 two subtypes : class ia , in which bifunctional small molecules forge 
 new intermolecular interactions , such as heterodimerization , that 
 in turn elicits a downstream biological response ( typically a gain 
 of function or increase in a background rate ) ; and class ib , where 
 two termini of the bifunctional small molecule interact with the same 
 target poi , such as in site - specific irreversible dye labeling or 
 covalent enzyme inactivation ( figure 1 ) . we stress that although applications that stem from 
 both subclasses are diverse and far - reaching , they are unified by 
 the elegant simplicity of a design based upon proximity promoting 
 a response / interaction . beyond cid - based implementation of tethered 
 probes , recent examples 
 in the first subclass include proteasome targeting , t cell activity control , antibody recruitment , and development of hybrid pharmaceuticals . the second subclass is represented by multifunctional small - molecule - derived 
 conjugates that function as potent and specific inhibitors , biosensors , reporters , and protein - activity modulators . we focus on the following latest developments exploiting small - molecule 
 scaffolding to elicit gain - of - function responses . we draw the reader s 
 attention to recent excellent and comprehensive accounts of cid - driven 
 biological applications and other approaches 
 utilizing small - molecule - based tethering platforms . proteolysis targeting chimeras ( protacs ) are bifunctional small 
 molecules that induce degradation of their target poi . protacs work by a validated mechanism in which 
 one pole of the protac binds to the poi while the other protac pole 
 is a recognition unit that recruits a specific e3 ligase . the proximity 
 of the ligase and target poi is sufficient to elicit polyubiquitination 
 of the poi , marking the target out for proteasomal degradation ( figure 2 ) . the original e3 
 ligase recognition pole was a functionalized peptide that specifically 
 recruits the von hippel the reliance 
 on peptide - based ligands was a significant stumbling block to the 
 broad applicability of protacs because peptides suffer from low cell 
 permeability and susceptibility to proteases , among other drawbacks . recently , vhl - specific 
 nonpeptide small - molecule ligands have been shown to function as the 
 e3 ligase recognition pole . most impressively , 
 each heterobifunctional all - small - molecule next - generation protac 
 can facilitate multiple protein - degradation turnovers , resulting in 
 > 90% knockdown  a result on par with complementary genetic 
 methods 
 such as shrna- and crispr / cas9-assisted gene knockdowns and knockouts . although many strategies for selective protein degradation of artificial 
 pois have been described , protacs remain 
 one of the most promising for drug discovery because they have been 
 proven to work on a variety of endogenous target pois . the tethered bifunctional 
 protac unites the poi with an e3 ubiquitin ligase , allowing for an 
 e2-mediated transfer of ubiquitin ( ub ) . inset : nonpeptide - derived 
 protacs that can induce targeted degradation of the nuclear estrogen - related 
 receptor alpha ( top ) and the serine / threonine kinase ripk2 ( bottom ) . t cells engineered 
 to express chimeric antigen receptors ( cars ) have proven effective 
 against refractory b cell cancers in clinical trials . despite this promise , sporadic toxicity has been observed 
 due to off - target killing of healthy cells . car  in which the 
 intracellular portion is expressed as two separate polypeptides that 
 can coalesce in the presence of a bifunctional small molecule  was 
 envisioned ( figure 3 ) . in the absence of the small molecule , the antigen receptor is defunct , and the t cells can not function effectively , 
 even in the presence of their cognate antigen . however , addition of 
 the small - molecule dimerizers allows the chimeric receptor to regain 
 function , and only in the presence of the antigen , are t cells activated . , 
 was shown to be able to elicit small - molecule - titratable tumor cell 
 killing and was tolerated by mice . on - switch cars are built upon a binary 
 logic gate system in which 
 simultaneous presence of tumor antigen and cid - based small molecule 
 dimerizer ( e.g. , rapamycin analogue ap21967 ) triggers t cell activation . our next example shows 
 how tethering can be equally effective at engendering a gain of function 
 by bringing two different cell types together . importantly , although the arena in which the events play 
 out is vastly different from those above , the foundational chemical 
 principles remain the same . synthetic antibody mimics ( syams ) use 
 a bifunctional small - molecule that can bind to both a cell - surface 
 membrane antigen , selectively overexpressed in prostate cancer cells , 
 and an effector domain that is present in immune cells , fc gamma receptors 
 ( fcrs ) ( figure 4 ) . thus , syams initiate selective phagocytosis 
 of the target cancer cells while avoiding noncancerous cells . the 
 advantages of syams over antibody therapies illustrate the need for 
 contextually relevant comparisons : for instance , although syams are 
 much larger than conventional small - molecule therapeutics , they are 
 only 5% of the molecular weight of an antibody ( many of which are 
 also used or in trials to treat various tumors ) , allowing for better 
 solid tumor penetration . many cell types also coexpress both activating 
 and inhibitory fcrs , both of which can be bound by antibodies . on the other hand , syams only bind type i ( activator ) fcr , 
 and hence are capable of selectively initiating immune - mediated toxicity . the tethered bifunctional syams unite disease - relevant targets 
 ( cancer cells / viral and bacteria pathogens ) and immune cells by binding 
 simultaneously to a specific membrane antigen and fc gamma receptor 
 i. this prompts immune clearance of the targets . inset , the third - generation 
 syam - p3 comprising a pair of cp33 , the fcgri binding motif , and a 
 pair of prostate - specific antigen binding motifs . interestingly , on - switch cars and syams show how a similar 
 problem  how 
 to harness immune cells for targeted therapeutic value  can 
 be tackled in two different ways while still using class ia tethering 
 as a core concept . in on - switch cars , intracellular tethering relays 
 signals in an engineered immune cell , whereas in syams , extracellular 
 tethering choreographs the physical interaction between the host s 
 immune cells and the intended target cell . this comparison thus serves 
 as testament to the versatility of bifunctional small molecules in 
 terms of their mechanism and applicability across various locales 
 and contexts . most class ia systems have no class ii counterpart 
 because the 
 small molecule must behave as a scaffold for a sustained period to 
 allow a response to occur . the recently demonstrated recyclability 
 of protacs allows for low dosage while 
 maintaining high efficacy , a benefit which is also conferred by class 
 ii strategies ( vide infra ) . in the case of protacs , 
 high efficacy at low dosage is possible because the protac proceeds 
 through noncovalently linked intermediates and elicits a gain of function 
 ( degradation ) . the latter highlights an important  win  
 for bifunctional molecules over single - site binders ( e.g. , inhibitors ) , 
 because small - molecule inhibitors typically require 50% or more target - site 
 occupancy to be effective . on the down 
 side , given the logistical issues with marshaling complex organizations , 
 intricate design is required to achieve success of class ia systems . furthermore , how such complex small molecules behave in vivo in terms of stability , tolerance , and off - target spectrum  which 
 is likely a function of both the poles and the linkers even if there is little problem with 
 chemical stability , the pathways targeted are intrinsically complex and future work must also seek to establish a deeper understanding 
 of the generality , scope , and shortcomings in various cell and tissue 
 types . encouragingly , protacs have recently shown efficacy in various 
 mouse tissues and early cid molecules 
 have also proven effective in mouse models . hopefully , the coming 
 years will see more data evaluating utility of bifunctional small - molecule 
 ligands in animals . the pressing need 
 to monitor changes in the abundance as well as spatiotemporal patterns 
 of small - molecule analytes in a wealth of applications has spurred 
 on the development of numerous sophisticated biosensors . several such biosensors exploiting tethered heterotrifunctional 
 small molecules have emerged over the past decade . in a recent invention 
 termed luciferase - based indicators of drugs ( lucids ) , a low - cost point - of - care 
 drug sensor is patterned using a three - component fusion protein : snap tag ( a 20 kda polypeptide that 
 binds covalently to small molecules functionalized with a benzylguanine 
 unit ) , nanoluciferase ( which engages 
 in a chemiluminescent reaction with its substrate furimazine ) , and the poi ( which binds to the drug in question ) . tethered trifunctional small molecules are custom - designed to recognize 
 each piece of the three - component fusion protein : a benzylguanine 
 motif that covalently binds to the snap tag , a fluorophore to permit 
 bioluminescence resonance energy transfer ( bret ) with nanoluciferase , and a noncovalent ligand specific 
 to the poi . covalent binding of snap tag to the trifunctional small 
 molecule and ensuing proximity - assisted noncovalent association of 
 the specific ligand on the opposite pole of the trifunctional small 
 molecule to the poi facilitates bret between the fluorophore and nanoluciferase 
 ( figure 5 ) . upon addition 
 of a free test ligand that competes with the tethered small molecule 
 for binding to the poi , the noncovalent interaction between the tethered 
 pole and the poi breaks , lowering the bret ratio . this system renders 
 the bret ratio dependent upon test ligand concentration and presents 
 a useful platform for real - time interrogations into dose - dependent 
 fluxes of therapeutics in blood samples . the ratiometric measurement 
 sidesteps some of the generic issues common to biosensors , affording the method an elegant simplicity of 
 use as a portable system for field testing . however , masterminding 
 these kinds of biosensors demands a great deal of protein engineering 
 to solve a complex three - component problem . additionally , this method 
 is currently limited to pois for which established reversible ligands 
 exist that , despite being chemically modified to accommodate a linker , 
 can bind a site on the poi that is regulated by binding of the test 
 ligand , e.g. , trimethoprim ( tmp ) to dihydrofolate reductase ( dhfr ) , cyclosporin a to cyclophilin a , and benzenesulfonamide derivatives to human carbonic anhydrase 
 ii ( hca ) . this strategy will only be 
 most accurate to titer concentrations of ligands that bind reversibly 
 to the target site ( arguably the most prevalent class of ligands available ) , and careful considerations must be taken if 
 the test ligand has long residence times on its target 
 poi to ensure complete equilibration . tethered tmp binding to dhfr ( poi ) 
 enables bret between 
 the nano - luc enzyme and cy3 dye . molecular switches have been reported in which a freely diffusible 
 effector protein sterically regulates binding of a functionalized 
 ligand to its poi . the setup of this 
 platform is similar to lucids in that 
 a covalent interaction between snap tag and a benzylguanine motif 
 orchestrates noncovalent assembly on a sophisticated fusion protein . in this instance , however , the engineered small - molecule probe contains 
 binding sites for the fusion protein as well as a binding site for 
 an exogenous protein ( figure 6a ) . the ligand to the exogenous protein and the ligand to 
 the poi within the fusion protein system are juxtaposed such that 
 addition of the exogenous protein and accompanying formation of a 
 new protein  ligand complex prevents the poi from binding to 
 the tethered ligand . this intervention provides a dynamic molecular 
 switch that is controlled by the concentration of the effector protein . 
 in a proof - of - principle example of this chemical ligand - associated 
 steric hindrance ( clash ) method , proximity - directed 
 control of the activity of luciferase fused to snap tag was demonstrated 
 ( figure 6a ) . in the 
 ground state , a multifunctional conglomerate is created , in which 
 luciferase is covalently linked to coelenteramide ( a reversible inhibitor ) via conjugation to the snap tag . the coelenteramide 
 pole is also functionalized with biotin . in the absence of streptavidin ( a tetrameric protein which binds 
 to biotin with high affinity ) , the coelenteramide pole efficiently 
 binds and inhibits luciferase . upon addition of streptavidin , the 
 coelenteramide inhibitor can no longer bind luciferase , triggering 
 luciferase bioluminescence ( figure 6a ) . this trifunctional 
 tethered manifold was further extended to incorporate a cy3 fluorophore between the snap tag and the biotin which enables 
 bret with nanoluciferase in the ground state ( figure 6b ) . in this instance , the fluorophore was 
 brought into contact with luciferase using hca and benzenesulfonamide . 
 by also appending biotin to complete the tetra - functional tethered 
 small - molecule arrangement made up of benzylguanine , cy3 , benzenesulfonamide , 
 and biotin , it was found that the interaction between hca and benzenesulfonamide 
 was prevented by streptavidin in a dose - dependent manner . a 
 potential drawback of the above strategy is that an exogenous protein 
 ( e.g. , streptavidin ) is required to effect a response . this set up 
 is arguably more amenable to the detection of a particular protein 
 rather than serving as a tunable molecular switch since protein expression 
 level is less easily controlled and can be changed with less precision 
 in terms of both timing and concentration than small - molecule ligand 
 addition , especially in cells and whole organisms . to enable more 
 precise control of the response , a four - component fusion protein comprising 
 an acetylcholine esterase ( ache ) , snap , and clip ( a 20 kda protein tag that 
 reacts with benzyl cytosine ) domains , 
 and a membrane - bound hca was conceived ( figure 6c ) . a tethered small - molecule array was constructed 
 in which edrophonium ( ligand to ache ) and benzenesulfonamide ( ligand 
 to hca ) were joined to a benzylguanine motif ( irreversible binder 
 to snap ) via a linker containing a cy5 fluorophore . the clip domain of the four - component fusion protein expressed 
 at the cell surface was prelabeled with a cy3 fluorophore through 
 the use of a benzylcytosine ( irreversible ligand to clip)-functionalized 
 cy3 probe ( figure 6c ) . in the absence of tacrine ( an ache inhibitor formerly used to 
 treat alzheimer s disease ) , intramolecular 
 binding of the tethered edrophonium to ache keeps the system in a 
 low - fret state while the snap domain functions as a hinge point through 
 which covalent ligand , the system allows the 
 tethered benzenesulfonamide to interact with hca ( figure 6c ) , switching to a high - fret 
 state as a result of donor ( cy3 ) to acceptor ( cy5 ) interaction . tacrine 
 induction and removal , respectively , enables the tethered ligand to 
 reversibly toggle between the binding of tacrine and edrophonium to 
 ache , which corresponds to high- vs low - fret states , respectively . this elegant strategy demonstrates the power of multiple intramolecular 
 tethers that can be fabricated in situ at the cell 
 surface and controlled by a simple change in conditions . however , 
 the level of complexity of the system is high , and successful examples 
 have only been demonstrated with extracellular and cell - free systems . clash 
 enables on / off switching of poi activity by making use of 
 two ligands with mutually exclusive binding to poi . ligand interaction that is disrupted 
 by an effector biomolecule [ streptavidin in ( a ) and ( b ) ] or small 
 molecule [ tacrine in ( c ) ] that competes with the ligand within the 
 tethered array that initially binds poi . in ( b ) , only the ground state 
 in the absence of streptavidin is shown . poi is exemplified by nanoluciferase 
 ( a ) and hca ( b , c ) . glycan 
 alterations at cell surfaces are responsible for many essential biological 
 functions , such as antigen presentation , cell - to - cell communication , 
 and cell differentiation and migration . it is thus critically 
 important to understand the makeup of the cell - surface glycome . many 
 methods to visualize global glycosylation at the plasma membrane have 
 been reported ; however , chemical tethering has 
 recently allowed individual membrane protein glycosylation events 
 to be identified . the approach uses a 
 dna aptamer that can bind specific cell - surface 
 targets . the aptamer is modified with biotin ( or a small - molecule 
 fluorophore ) at one terminus and cyclooctyne at the other . cells are 
 first pretreated with peracetylated n - azidoacetylneuraminic 
 acid ( sianaz ) , which is incorporated into glycoproteins by endogenous 
 glycosylation pathways . when the aptamer binds to its target receptor 
 at the cell surface , provided the target itself has been sialylated 
 with sianaz , accompanying enhanced local concentration primes rapid 
 covalent conjugation of the cyclooctyne and the azide group via copper - free 
 click coupling ( figure 7 ) . using this approach , ptk7 , an important 
 receptor in the wnt signaling pathway , was identified as a novel sialylated receptor . although this method 
 promises exciting future expansion and applications , the authors provide 
 some evidence to suggest that in certain applications , the cyclooctyne 
 ligand can undergo reaction with nucleophilic residues leading to relatively high background . scaffolding 
 via an aptamer allows glycoprotein - specific fluorophore 
 targeting against the backdrop of metabolically sianaz - labeled cell - surface 
 glycans . proximity enables strain - promoted click coupling between 
 cyclooctyne tethered to aptamer and the azidosugar - labeled receptor . the demand for targeted small - molecule 
 inhibitors or ligands that 
 can selectively alter poi activity without affecting the entire proteome 
 has continued to grow over the years . this need 
 is magnified because there are many protein targets with high homology , 
 but distinct differences in their biological functions . identification of specific ligands for these 
 homologous targets remains a significant challenge . by incorporating a genetically encoded unnatural amino acid on the poi , a new bioorthogonal ligand tethering 
 ( bolt ) approach was designed which is capable of regulating the desired 
 poi with unparalleled rapidity and selectivity . the unnatural amino 
 acid within the poi reacts with one pole of a bifunctional inhibitor , 
 while the second pole within the tether functions as a low - affinity 
 ligand . many proteins may be able to bind the low - affinity ligand , 
 yet only the engineered mutant poi can be inactivated irreversibly 
 due to formation of a stable ligand - poi complex . the binding enhancement 
 conferred by the covalent interaction is most pronounced when a ligand 
 concentration below kd is used , such that 
 the ligand has low nontemplated occupancy , but irreversible chemistry 
 drives saturation of the target poi . the utility of this method was 
 illustrated by rapid and specific inhibition of specific mek isozymes featuring complementary unnatural amino acids . a further development uses photochemistry to allow toggling between 
 the inhibited and uninhibited states . this work builds upon prior 
 art in which photochromic tethered ligands ( ptls ) have been used to 
 elicit optogenetic control of living systems . a recent review thoughtfully 
 summarizes the design and applications of tethered small - molecule 
 photoswitches in live cells and animals . the resulting on / off approach called  photo - bolt  
 uses a cis trans photoisomerizable 
 linker between the two poles of the ligand , affording dynamic optochemical 
 control ( figure 8) . 
 in the photo - bolt platform , when the 
 linker is in the trans conformation , one pole of the bifunctional small molecule is covalently 
 conjugated to the unnatural amino acid , and the other pole noncovalently 
 binds to the active site of the poi , resulting in inhibition . inhibition 
 is reversed by illumination of the cells at a wavelength that initiates trans to cis isomerization of the linker . 
 in the cis isomer , the spatial arrangement of the 
 poles is changed such that the noncovalent - binding pole of the ligand 
 is no longer able to interact with the poi and activity is restored . initial proof - of - principle work has demonstrated that bolt and its 
 derivatives are effective in targeting a specific enzyme over a number 
 of closely related analogues . the reliance on unnatural amino acids 
 is liberating as there is no need for large domain fusions that may 
 affect protein function , but also potentially restrictive , because 
 a readily functionalizable residue must be found in proximity to the 
 target ligand binding site that must preserve activity even ( in the 
 case of photo - bolt ) when functionalized with one geometrical isomer 
 of the bolt ligand . one interesting question is how distance from 
 the target binding site and linker length affect the benefit conferred 
 by intermolecular binding . depending on the importance of these parameters , 
 one could envision using a separate binding domain ( e.g. , snap ) to 
 anchor the ligand to the poi in a similar way to ibolt . the bifunctional small - molecule probe undergoes 
 an inverse - electron demand diels  alder ( iedda)/retro diels  alder 
 reaction with the unnatural amino acid on the poi to form a covalent 
 linkage . both proximity - directed ligation and bolt use enhanced 
 local concentrations 
 of a semispecific probe to direct a specific interaction to a desired 
 point of origin . clearly for many scenarios however , it remains to be seen how effective systems like 
 bolt will be in more complex settings . for instance , when the target 
 poi can form hetero - oligomers with other isozymes that can interact 
 with the noncovalent - binding pole of the bifunctional probe , specificity 
 will likely be diminished . in the event that interprotein interactions 
 dominate , inhibition of the undesired isoform / protein may result . judicious choice of a linker length / unnatural amino acid position 
 that favors intramolecular interactions may circumvent this issue . the site - specific noncovalent labeling of dhfr - fusion 
 proteins with tmp - conjugated fluorescent dyes and analogous small - molecule 
 dye labeling methods are widely used . in the case 
 of dhfr and tmp , the assembly exploits the selective , noncovalent 
 binding of the tmp ligand to escherichia coli as 
 opposed to human dhfr , which results in a protein  ligand complex 
 with a dissociative half - life on the order of 20 min . to increase the residence time of the ligand 
 on the target enzyme , this platform has been recently amended such 
 that a covalent ( essentially permanent ) bond between the tmp and dhfr 
 is formed ( figure 9 ) . this required 
 single modifications to the ligand and protein : synthetic derivatization 
 of the tmp - conjugated dye to incorporate an acrylamide appendage , 
 and a point mutation ( k28c ) within dhfr . binding affinity of the k28c - dhfr 
 to tmp is similar to that of the wild type , indicating that the mutant 
 enzyme retains its affinity for tmp ; however , when the acrylamide - functionalized 
 ligand is used , a proximity - induced michael addition covalently links 
 the nucleophilic c28 residue on the mutant dhfr with the ligand . a 
 related development utilizing intramolecular tethering for covalent - dye 
 labeling makes use of fluorophore - binding peptides , called fluorettes 
 ( figure 10 ) . in this case , a reactive cysteine residue is 
 built into the linker region of the fusion protein construct made 
 up of a fluorette fused to the poi . the tethered small molecule is 
 designed with an -halocarbonyl function appended to a texasred - derived 
 fluorophore known to bind reversibly to the specific fluorette . the rapid noncovalent interaction between the 
 fluorophore and the fluorette templates a covalent reaction between 
 the -halocarbonyl motif on the tether and the proximal cysteine 
 on the linker attached to the target poi . covalent capture enables 
 stable dye incorporation through an acylamide 
 appendage that can conjugate with the engineered cysteine k28c - dhfr 
 mutant fused to poi . the crux of the method is that typically covalent interactions 
 occur slower than noncovalent associations . should a relatively rapid 
 noncovalent association facilitate covalent bond formation , the rate 
 enhancement relative to nonspecific covalent bond formation will allow 
 a specific covalent bond forming event to occur . thus , the best systems 
 will have low intrinsic reactivity of the reactive pole , or  warhead  . 
 obviously , for this process to occur a nucleophilic residue ( usually 
 cysteine ) must be in close enough proximity to the ligand when bound 
 to its target to form a covalent bond to the acrylamide moiety . similar 
 considerations have been echoed by the pharmaceutical communities 
 as attempts are made to develop irreversible inhibitors from known 
 reversible binders . the acrylamide warhead demonstrated for the dhfr / tmp system is of 
 relatively low intrinsic reactivity / promiscuity , so its use will limit 
 off - target binding . it is thus ideally set to pair with trimethoprim , 
 which is a high - affinity , reversible , yet slow onset inhibitor of 
 dhfr . it is possible to increase the promiscuity of the reactive unit 
 appended to the ligand [ such as the -halocarbonyl in the fluorette 
 system ( figure 10 ) ] 
 to drive covalent attachment ; however , increasing reactivity will 
 typically elevate off - target binding , and will ultimately limit the 
 versatility of the method . clearly , such promiscuous reactive ligands 
 may not be kinetically compatible with slow onset inhibitor manifolds 
 either . similar ways to engender specificity have been achieved using 
 unnatural amino acids as in bolt for instance , and cyclooctyne such 
 as in proximity - enhanced aptamer ligation . one alternative solution 
 applicable to ligands with relatively long residence times on their 
 target is on - demand activation of a reactive functional group once 
 the recognition element has been bound to the poi and the excess washed 
 away . in this context , recently developed on - demand targeting or in situ unmasking ideas of proximity enhancement have appeared 
 in the literature ( vide infra ) . covalent capture concept 
 in stable dye incorporation using a fusion 
 peptide that bears a texas red fluorophore - binding sequence ( blue ) 
 and reactive cys residues . the pioneering lines of method development 
 within the class i framework over the recent decade have also highlighted 
 several general as well as specific challenges unique to individual 
 subclasses . both covalent and noncovalent modes of ligand binding are evident 
 in the examples illustrated above , but the decision as to which strategy 
 is better integrated in developing a new toolset is often not obvious . key aspects are the following : ( 1 ) reversible binders 
 can function catalytically allowing low ligand load while maintaining 
 high efficacy , whereas irreversible binders can not . ( 2 ) although typically assumed to be rapid , 
 binding equilibria of noncovalent ligands may be relatively 
 slowly established and occur in a complex , multistep process involving 
 enzyme conformational changes , especially for high - affinity binders . crystal structures of protein / ligand complexes will typically show 
 end - point conformations , and thus careful consideration of proximal 
 residues must be made especially if a class ib strategy is under consideration . ( 3 ) off - rates ( residence times ) of high - affinity reversible ligands 
 can also be prolonged , making washout of ligand prior to downstream 
 analysis , or sensing of analyte concentrations , require careful optimization . ( 4 ) covalent bond formation is typically slow ( detection 
 of a binding event may take minutes to hours ) , leading to time - dependent associations that need to be addressed 
 carefully to account for off - target effects and characterization of 
 ligand  protein interactions . ( 5 ) although not all off - target 
 interactions of irreversible target binders are necessarily covalent , 
 those that are will increase as a function of time . unlike for noncovalent 
 off - target effects , the covalent off - targets , once formed , necessitate 
 new protein synthesis to regain activity if target inactivation has 
 occurred . alternatively , if gain of function has occurred , degradation 
 coupled with new protein synthesis is required . given the above , we 
 finally stress that conversion of noncovalent binders to covalent 
 binders in a bid to assess on- and/or off - target binding should be 
 approached with caution . one 
 trivial , but nonetheless common issue is that for classes ia and b , 
 care must be taken to ensure that tethered macromolecules forged by 
 the multifunctional small - molecule scaffolds ( either via intermolecular 
 protein  protein tethering enabled by the probes , or intramolecular 
 protein  ligand tethering ) are able to maintain functionality . an arduous series of chemical syntheses ( or permuted polypeptides ) , 
 optimizations of linkers and ordering of individual functional groups 
 along the tethered multifunctional small - molecule array are often 
 prerequisites of many successful class i methods discussed above . 
 currently there are no clear predictive tools for achieving the optimal 
 design a priori . however , what has been less well 
 recognized is the fact that these considerations can also be used 
 to the researcher s advantage . in clash ( figure 6 ) , problems associated with steric repulsion have been turned on 
 their head by designing techniques based on steric - strain - directed 
 ligand binding and toggling . photo - bolt also utilizes rigidity - enhanced steric and stereoelectronic requirements 
 of intramolecular bond formation to its advantage ( figure 8) . one generally less attractive feature of systems 
 in which a bifunctional 
 ligand must interact with two independent binding sites is the prozone 
 or hook effect , wherein a drop in ternary 
 complex formation occurs at high ligand concentrations . this behavior 
 counteracts one of the key benefits of small molecules  their 
 predictable dose response . the hook effect occurs because once the 
 ligand is in excess relative to its target protein partners , there 
 is sufficient free ligand available for each of the two poles to bind 
 its respective protein target separately . although 
 this behavior has been helpful to model binding mechanisms in vitro , this complex operation confounds downstream biology , 
 especially in cells and whole organisms in which available small - molecule 
 ligand concentrations are not a simple parameter to control but often 
 dependent on pleiotropic factors . this problem is most applicable 
 to cases where both poles of the bifunctional probe interact reversibly 
 as in class ia systems discussed above . if one pole behaves as an 
 irreversible binder , the hook effect can be reduced , principally because 
 probe concentrations can be more readily controlled and excess can 
 be removed . on the other hand , the hook effect is less of an issue 
 if the ligand can behave catalytically as in next - generation protacs 
 because ligand concentrations can be minimized such that competition 
 for binding sites does not occur . finally , the hook effect may be 
 almost completely obviated in platforms such as bolt in which one 
 pole of the bifunctional probe reacts irreversibly and essentially 
 exclusively with its target site and the second binding site is also 
 on the same protein ( class ib systems ) . in terms of downstream applications , the majority of applications 
 using proximity - induced chemistry are limited by the complexity of 
 the molecules required to achieve their ultimate goal . thus , most 
 multicomponent tethered probes ( classes ia and ib)even those 
 currently at the cutting edge of this field  work well in cultured 
 cells , but much less information is available on how well they are 
 adaptable to applications in animal models or for human disease treatments . the molecular design inherently calls for 
 two poles that are each sufficiently complex to bind selectively to 
 a specific target poi , and a linker separating the poles . these prerequisites 
 render it challenging to create bifunctional small molecules that 
 adhere to basic drug design rules to optimize admet ( absorption , distribution , 
 metabolism , excretion , and toxicity ) properties , such as lipinski s rule of five . beyond the above considerations , increased size / complexity 
 of chimeric ligands may engender lower target specificity compared 
 to single - site binders . however , this proposition is not necessarily 
 true because the downstream application of single - site - binding ligands 
 and bifunctional molecules can be very different . single - site binders 
 often act as inhibitors of a specific enzyme , but dimerizers ( i.e. , 
 class ia ) induce gain of function through forcing protein it is thus possible that by setting up low - occupancy 
 binding conditions ( i.e. , less than stoichiometric target - binding ) , 
 even chimeras derived from promiscuous ligands can have highly specific 
 effects . this is because formation of the desired protein complex 
 will cause a large gain in activity even at low ligand occupancy , 
 whereas unintentional off - target binding will be low occupancy and 
 is unlikely to usher a gain of function . the multicomponent scaffolding 
 is the centerpiece of both classes 
 ia and ib probe design in which each of the components is typically 
 derived from known ligands . unfortunately , not all protein ligands 
 are amenable to functionalization , either because they lack a suitable 
 functionalizable appendage , or because chemical modification perturbs 
 function . thus , there are relatively few bona fide ligand  protein pairs directly amenable to class furthermore , most of the functionalizable protein ligands are actually 
 inhibitors of a specific enzyme , principally because protein binders 
 have traditionally been identified from activity modulation screens . this caveat often limits downstream chemical biology applications 
 because protein targets are mostly restricted to those with an enzymatic 
 function , but that activity will be 
 inhibited as consequence of ligand binding . various methods have been 
 used to combat this deficiency , including the use of artificial domains 
 ( classes ia and ib ) such as dhfr . these considerations are not applicable 
 if a pole can be found that interacts with a site that is not functionally 
 coupled to the target enzyme s activity ; e.g. , the target protein 
 is part of a complex . for instance , in the case of protacs , catalytic 
 chemistry ( ubiquitination ) is carried out by the e2 ligase , which 
 is complexed to the target of the protac , an e3 ligase . more 
 recently , drug - screening methods that append macromolecular barcodes 
 onto small - molecule ligands have become widespread . these innovations provide a goldmine of potential small - molecule 
 ligands ( not necessarily inhibitors ) selective for a particular poi 
 ( not necessarily those with enzymatic functions ) that must be tolerable 
 to functionalization to appear as a hit . on the bright side , for proof 
 of principle , genetically encodable protein tags  such as snap , clip , dhfr , halo , and prime  can greatly extend the scope and generality of all 
 methodologies , provided the researcher is prepared to study ectopic 
 proteins and evaluate that the tag does not perturb protein function 
 drastically . the ease with which knockdowns / knockouts or inducible 
 systems can now be generated additionally provides 
 opportunities to study these fusion pois against the null background 
 or with minimal perturbation to the endogenous organizations . with 
 the advent of engineered immune cells for targeted therapy , there is also a distinct possibility that these artificial domains 
 could be used in  real - life  applications . we focus on the following latest developments exploiting small - molecule 
 scaffolding to elicit gain - of - function responses . we draw the reader s 
 attention to recent excellent and comprehensive accounts of cid - driven 
 biological applications and other approaches 
 utilizing small - molecule - based tethering platforms . proteolysis targeting chimeras ( protacs ) are bifunctional small 
 molecules that induce degradation of their target poi . protacs work by a validated mechanism in which 
 one pole of the protac binds to the poi while the other protac pole 
 is a recognition unit that recruits a specific e3 ligase . the proximity 
 of the ligase and target poi is sufficient to elicit polyubiquitination 
 of the poi , marking the target out for proteasomal degradation ( figure 2 ) . the original e3 
 ligase recognition pole was a functionalized peptide that specifically 
 recruits the von hippel the reliance 
 on peptide - based ligands was a significant stumbling block to the 
 broad applicability of protacs because peptides suffer from low cell 
 permeability and susceptibility to proteases , among other drawbacks . recently , vhl - specific 
 nonpeptide small - molecule ligands have been shown to function as the 
 e3 ligase recognition pole . . most impressively , 
 each heterobifunctional all - small - molecule next - generation protac 
 can facilitate multiple protein - degradation turnovers , resulting in 
 > 90% knockdown  a result on par with complementary genetic 
 methods 
 such as shrna- and crispr / cas9-assisted gene knockdowns and knockouts . although many strategies for selective protein degradation of artificial 
 pois have been described , protacs remain 
 one of the most promising for drug discovery because they have been 
 proven to work on a variety of endogenous target pois . the tethered bifunctional 
 protac unites the poi with an e3 ubiquitin ligase , allowing for an 
 e2-mediated transfer of ubiquitin ( ub ) . inset : nonpeptide - derived 
 protacs that can induce targeted degradation of the nuclear estrogen - related 
 receptor alpha ( top ) and the serine / threonine kinase ripk2 ( bottom ) . t cells engineered 
 to express chimeric antigen receptors ( cars ) have proven effective 
 against refractory b cell cancers in clinical trials . despite this promise , sporadic toxicity has been observed 
 due to off - target killing of healthy cells . thus , to design precision - controlled 
 therapeutic t cells , a  split car  in which the 
 intracellular portion is expressed as two separate polypeptides that 
 can coalesce in the presence of a bifunctional small molecule  was 
 envisioned ( figure 3 ) . in the absence of the small molecule , the antigen receptor is defunct , and the t cells can not function effectively , 
 even in the presence of their cognate antigen . however , addition of 
 the small - molecule dimerizers allows the chimeric receptor to regain 
 function , and only in the presence of the antigen , are t cells activated . , 
 was shown to be able to elicit small - molecule - titratable tumor cell 
 killing and was tolerated by mice . on - switch cars are built upon a binary 
 logic gate system in which 
 simultaneous presence of tumor antigen and cid - based small molecule 
 dimerizer ( e.g. , rapamycin analogue ap21967 ) triggers t cell activation . our next example shows 
 how tethering can be equally effective at engendering a gain of function 
 by bringing two different cell types together . importantly , although the arena in which the events play 
 out is vastly different from those above , the foundational chemical 
 principles remain the same . synthetic antibody mimics ( syams ) use 
 a bifunctional small - molecule that can bind to both a cell - surface 
 membrane antigen , selectively overexpressed in prostate cancer cells , 
 and an effector domain that is present in immune cells , fc gamma receptors 
 ( fcrs ) ( figure 4 ) . thus , syams initiate selective phagocytosis 
 of the target cancer cells while avoiding noncancerous cells . the 
 advantages of syams over antibody therapies illustrate the need for 
 contextually relevant comparisons : for instance , although syams are 
 much larger than conventional small - molecule therapeutics , they are 
 only 5% of the molecular weight of an antibody ( many of which are 
 also used or in trials to treat various tumors ) , allowing for better 
 solid tumor penetration . many cell types also coexpress both activating 
 and inhibitory fcrs , both of which can be bound by antibodies . 
 on the other hand , syams only bind type i ( activator ) fcr , 
 and hence are capable of selectively initiating immune - mediated toxicity the tethered bifunctional syams unite disease - relevant targets 
 ( cancer cells / viral and bacteria pathogens ) and immune cells by binding 
 simultaneously to a specific membrane antigen and fc gamma receptor 
 i. this prompts immune clearance of the targets . inset , the third - generation 
 syam - p3 comprising a pair of cp33 , the fcgri binding motif , and a 
 pair of prostate - specific antigen binding motifs . interestingly , on - switch cars and syams show how a similar 
 problem  how 
 to harness immune cells for targeted therapeutic value  can 
 be tackled in two different ways while still using class ia tethering 
 as a core concept . in on - switch cars , intracellular tethering relays 
 signals in an engineered immune cell , whereas in syams , extracellular 
 tethering choreographs the physical interaction between the host s 
 immune cells and the intended target cell . this comparison thus serves 
 as testament to the versatility of bifunctional small molecules in 
 terms of their mechanism and applicability across various locales 
 and contexts . most class ia systems have no class ii counterpart 
 because the 
 small molecule must behave as a scaffold for a sustained period to 
 allow a response to occur . the recently demonstrated recyclability 
 of protacs allows for low dosage while 
 maintaining high efficacy , a benefit which is also conferred by class 
 ii strategies ( vide infra ) . in the case of protacs , 
 high efficacy at low dosage is possible because the protac proceeds 
 through noncovalently linked intermediates and elicits a gain of function 
 ( degradation ) . the latter highlights an important  win  
 for bifunctional molecules over single - site binders ( e.g. , inhibitors ) , 
 because small - molecule inhibitors typically require 50% or more target - site 
 occupancy to be effective . on the down 
 side , given the logistical issues with marshaling complex organizations , 
 intricate design is required to achieve success of class ia systems . 
 furthermore , how such complex small molecules behave in vivo in terms of stability , tolerance , and off - target spectrum  which 
 is likely a function of both the poles and the linkers  still 
 needs to be fully evaluated . even if there is little problem with 
 chemical stability , the pathways targeted are intrinsically complex and future work must also seek to establish a deeper understanding 
 of the generality , scope , and shortcomings in various cell and tissue 
 types . encouragingly , protacs have recently shown efficacy in various 
 mouse tissues and early cid molecules 
 have also proven effective in mouse models . hopefully , the coming 
 years will see more data evaluating utility of bifunctional small - molecule 
 ligands in animals . the pressing need 
 to monitor changes in the abundance as well as spatiotemporal patterns 
 of small - molecule analytes in a wealth of applications has spurred 
 on the development of numerous sophisticated biosensors . several such biosensors exploiting tethered heterotrifunctional 
 small molecules have emerged over the past decade . in a recent invention 
 termed luciferase - based indicators of drugs ( lucids ) , a low - cost point - of - care 
 drug sensor is patterned using a three - component fusion protein : snap tag ( a 20 kda polypeptide that 
 binds covalently to small molecules functionalized with a benzylguanine 
 unit ) , nanoluciferase ( which engages 
 in a chemiluminescent reaction with its substrate furimazine ) , and the poi ( which binds to the drug in question ) . 
 tethered trifunctional small molecules are custom - designed to recognize 
 each piece of the three - component fusion protein : a benzylguanine 
 motif that covalently binds to the snap tag , a fluorophore to permit 
 bioluminescence resonance energy transfer ( bret ) with nanoluciferase , and a noncovalent ligand specific 
 to the poi . covalent binding of snap tag to the trifunctional small 
 molecule and ensuing proximity - assisted noncovalent association of 
 the specific ligand on the opposite pole of the trifunctional small 
 molecule to the poi facilitates bret between the fluorophore and nanoluciferase 
 ( figure 5 ) . upon addition 
 of a free test ligand that competes with the tethered small molecule 
 for binding to the poi , the noncovalent interaction between the tethered 
 pole and the poi breaks , lowering the bret ratio . this system renders 
 the bret ratio dependent upon test ligand concentration and presents 
 a useful platform for real - time interrogations into dose - dependent 
 fluxes of therapeutics in blood samples . the ratiometric measurement 
 sidesteps some of the generic issues common to biosensors , affording the method an elegant simplicity of 
 use as a portable system for field testing . however , masterminding 
 these kinds of biosensors demands a great deal of protein engineering 
 to solve a complex three - component problem . additionally , this method 
 is currently limited to pois for which established reversible ligands 
 exist that , despite being chemically modified to accommodate a linker , 
 can bind a site on the poi that is regulated by binding of the test 
 ligand , e.g. , trimethoprim ( tmp ) to dihydrofolate reductase ( dhfr ) , cyclosporin a to cyclophilin a , and benzenesulfonamide derivatives to human carbonic anhydrase 
 ii ( hca ) . this strategy will only be 
 most accurate to titer concentrations of ligands that bind reversibly 
 to the target site ( arguably the most prevalent class of ligands available ) , and careful considerations must be taken if 
 the test ligand has long residence times on its target 
 poi to ensure complete equilibration . tethered tmp binding to dhfr ( poi ) 
 enables bret between 
 the nano - luc enzyme and cy3 dye . molecular switches have been reported in which a freely diffusible 
 effector protein sterically regulates binding of a functionalized 
 ligand to its poi . the setup of this 
 platform is similar to lucids in that 
 a covalent interaction between snap tag and a benzylguanine motif 
 orchestrates noncovalent assembly on a sophisticated fusion protein . 
 in this instance , however , the engineered small - molecule probe contains 
 binding sites for the fusion protein as well as a binding site for 
 an exogenous protein ( figure 6a ) . the ligand to the exogenous protein and the ligand to 
 the poi within the fusion protein system are juxtaposed such that 
 addition of the exogenous protein and accompanying formation of a 
 new protein  ligand complex prevents the poi from binding to 
 the tethered ligand . this intervention provides a dynamic molecular 
 switch that is controlled by the concentration of the effector protein . 
 in a proof - of - principle example of this chemical ligand - associated 
 steric hindrance ( clash ) method , proximity - directed 
 control of the activity of luciferase fused to snap tag was demonstrated 
 ( figure 6a ) . in the 
 ground state , a multifunctional conglomerate is created , in which 
 luciferase is covalently linked to coelenteramide ( a reversible inhibitor ) via conjugation to the snap tag . the coelenteramide 
 pole is also functionalized with biotin . in the absence of streptavidin ( a tetrameric protein which binds 
 to biotin with high affinity ) , the coelenteramide pole efficiently 
 binds and inhibits luciferase . upon addition of streptavidin , the 
 coelenteramide inhibitor can no longer bind luciferase , triggering 
 luciferase bioluminescence ( figure 6a ) . this trifunctional 
 tethered manifold was further extended to incorporate a cy3 fluorophore between the snap tag and the biotin which enables 
 bret with nanoluciferase in the ground state ( figure 6b ) . in this instance , the fluorophore was 
 brought into contact with luciferase using hca and benzenesulfonamide . 
 by also appending biotin to complete the tetra - functional tethered 
 small - molecule arrangement made up of benzylguanine , cy3 , benzenesulfonamide , 
 and biotin , it was found that the interaction between hca and benzenesulfonamide 
 was prevented by streptavidin in a dose - dependent manner . a 
 potential drawback of the above strategy is that an exogenous protein 
 ( e.g. , streptavidin ) is required to effect a response . this set up 
 is arguably more amenable to the detection of a particular protein 
 rather than serving as a tunable molecular switch since protein expression 
 level is less easily controlled and can be changed with less precision 
 in terms of both timing and concentration than small - molecule ligand 
 addition , especially in cells and whole organisms . to enable more 
 precise control of the response , a four - component fusion protein comprising 
 an acetylcholine esterase ( ache ) , snap , and clip ( a 20 kda protein tag that 
 reacts with benzyl cytosine ) domains , 
 and a membrane - bound hca was conceived ( figure 6c ) . a tethered small - molecule array was constructed 
 in which edrophonium ( ligand to ache ) and benzenesulfonamide ( ligand 
 to hca ) were joined to a benzylguanine motif ( irreversible binder 
 to snap ) via a linker containing a cy5 fluorophore . the clip domain of the four - component fusion protein expressed 
 at the cell surface was prelabeled with a cy3 fluorophore through 
 the use of a benzylcytosine ( irreversible ligand to clip)-functionalized 
 cy3 probe ( figure 6c ) . in the absence of tacrine ( an ache inhibitor formerly used to 
 treat alzheimer s disease ) , intramolecular 
 binding of the tethered edrophonium to ache keeps the system in a 
 low - fret state while the snap domain functions as a hinge point through 
 which covalent ligand  protein conjugation is achieved . when 
 edrophonium is displaced from ache by tacrine , the system allows the 
 tethered benzenesulfonamide to interact with hca ( figure 6c ) , switching to a high - fret 
 state as a result of donor ( cy3 ) to acceptor ( cy5 ) interaction . tacrine 
 induction and removal , respectively , enables the tethered ligand to 
 reversibly toggle between the binding of tacrine and edrophonium to 
 ache , which corresponds to high- vs low - fret states , respectively . this elegant strategy demonstrates the power of multiple intramolecular 
 tethers that can be fabricated in situ at the cell 
 surface and controlled by a simple change in conditions . however , 
 the level of complexity of the system is high , and successful examples 
 have only been demonstrated with extracellular and cell - free systems . clash 
 enables on / off switching of poi activity by making use of 
 two ligands with mutually exclusive binding to poi . ligand interaction that is disrupted 
 by an effector biomolecule [ streptavidin in ( a ) and ( b ) ] or small 
 molecule [ tacrine in ( c ) ] that competes with the ligand within the 
 tethered array that initially binds poi . in ( b ) , only the ground state 
 in the absence of streptavidin is shown . poi is exemplified by nanoluciferase 
 ( a ) and hca ( b , c ) . glycan 
 alterations at cell surfaces are responsible for many essential biological 
 functions , such as antigen presentation , cell - to - cell communication , 
 and cell differentiation and migration . it is thus critically 
 important to understand the makeup of the cell - surface glycome . many 
 methods to visualize global glycosylation at the plasma membrane have 
 been reported ; however , chemical tethering has 
 recently allowed individual membrane protein glycosylation events 
 to be identified . the approach uses a 
 dna aptamer that can bind specific cell - surface 
 targets . the aptamer is modified with biotin ( or a small - molecule 
 fluorophore ) at one terminus and cyclooctyne at the other . cells are 
 first pretreated with peracetylated n - azidoacetylneuraminic 
 acid ( sianaz ) , which is incorporated into glycoproteins by endogenous 
 glycosylation pathways . when the aptamer binds to its target receptor 
 at the cell surface , provided the target itself has been sialylated 
 with sianaz , accompanying enhanced local concentration primes rapid 
 covalent conjugation of the cyclooctyne and the azide group via copper - free 
 click coupling ( figure 7 ) . using this approach , ptk7 , an important 
 receptor in the wnt signaling pathway , was identified as a novel sialylated receptor . although this method 
 promises exciting future expansion and applications , the authors provide 
 some evidence to suggest that in certain applications , the cyclooctyne 
 ligand can undergo reaction with nucleophilic residues leading to relatively high background . scaffolding 
 via an aptamer allows glycoprotein - specific fluorophore 
 targeting against the backdrop of metabolically sianaz - labeled cell - surface 
 glycans . proximity enables strain - promoted click coupling between 
 cyclooctyne tethered to aptamer and the azidosugar - labeled receptor . the demand for targeted small - molecule 
 inhibitors or ligands that 
 can selectively alter poi activity without affecting the entire proteome 
 has continued to grow over the years . this need 
 is magnified because there are many protein targets with high homology , 
 but distinct differences in their biological functions . identification of specific ligands for these 
 homologous targets remains a significant challenge . by incorporating a genetically encoded unnatural amino acid on the poi , a new bioorthogonal ligand tethering 
 ( bolt ) approach was designed which is capable of regulating the desired 
 poi with unparalleled rapidity and selectivity . the unnatural amino 
 acid within the poi reacts with one pole of a bifunctional inhibitor , 
 while the second pole within the tether functions as a low - affinity 
 ligand . many proteins may be able to bind the low - affinity ligand , 
 yet only the engineered mutant poi can be inactivated irreversibly 
 due to formation of a stable ligand - poi complex . the binding enhancement 
 conferred by the covalent interaction is most pronounced when a ligand 
 concentration below kd is used , such that 
 the ligand has low nontemplated occupancy , but irreversible chemistry 
 drives saturation of the target poi . the utility of this method was 
 illustrated by rapid and specific inhibition of specific mek isozymes featuring complementary unnatural amino acids . a further development uses photochemistry to allow toggling between 
 the inhibited and uninhibited states . this work builds upon prior 
 art in which photochromic tethered ligands ( ptls ) have been used to 
 elicit optogenetic control of living systems . a recent review thoughtfully 
 summarizes the design and applications of tethered small - molecule 
 photoswitches in live cells and animals . the resulting on / off approach called  photo - bolt  
 uses a cis trans photoisomerizable 
 linker between the two poles of the ligand , affording dynamic optochemical 
 control ( figure 8) . 
 in the photo - bolt platform , when the 
 linker is in the trans conformation , one pole of the bifunctional small molecule is covalently 
 conjugated to the unnatural amino acid , and the other pole noncovalently 
 binds to the active site of the poi , resulting in inhibition . inhibition 
 is reversed by illumination of the cells at a wavelength that initiates trans to cis isomerization of the linker . 
 in the cis isomer , the spatial arrangement of the 
 poles is changed such that the noncovalent - binding pole of the ligand 
 is no longer able to interact with the poi and activity is restored . initial proof - of - principle work has demonstrated that bolt and its 
 derivatives are effective in targeting a specific enzyme over a number 
 of closely related analogues . the reliance on unnatural amino acids 
 is liberating as there is no need for large domain fusions that may 
 affect protein function , but also potentially restrictive , because 
 a readily functionalizable residue must be found in proximity to the 
 target ligand binding site that must preserve activity even ( in the 
 case of photo - bolt ) when functionalized with one geometrical isomer 
 of the bolt ligand . one interesting question is how distance from 
 the target binding site and linker length affect the benefit conferred 
 by intermolecular binding . depending on the importance of these parameters , 
 one could envision using a separate binding domain ( e.g. , snap ) to 
 anchor the ligand to the poi in a similar way to ibolt . the bifunctional small - molecule probe undergoes 
 an inverse - electron demand diels  alder ( iedda)/retro diels  alder 
 reaction with the unnatural amino acid on the poi to form a covalent 
 linkage . trans isomerization enables reversible activity modulation . both proximity - directed ligation and bolt use enhanced 
 local concentrations 
 of a semispecific probe to direct a specific interaction to a desired 
 point of origin . clearly for many scenarios however , it remains to be seen how effective systems like 
 bolt will be in more complex settings . for instance , when the target 
 poi can form hetero - oligomers with other isozymes that can interact 
 with the noncovalent - binding pole of the bifunctional probe , specificity 
 will likely be diminished . in the event that interprotein interactions 
 dominate , inhibition of the undesired isoform / protein may result . judicious choice of a linker length / unnatural amino acid position 
 that favors intramolecular interactions may circumvent this issue . the site - specific noncovalent labeling of dhfr - fusion 
 proteins with tmp - conjugated fluorescent dyes and analogous small - molecule 
 dye labeling methods are widely used . in the case 
 of dhfr and tmp , the assembly exploits the selective , noncovalent 
 binding of the tmp ligand to escherichia coli as 
 opposed to human dhfr , which results in a protein  ligand complex 
 with a dissociative half - life on the order of 20 min . to increase the residence time of the ligand 
 on the target enzyme , this platform has been recently amended such 
 that a covalent ( essentially permanent ) bond between the tmp and dhfr 
 is formed ( figure 9 ) . this required 
 single modifications to the ligand and protein : synthetic derivatization 
 of the tmp - conjugated dye to incorporate an acrylamide appendage , 
 and a point mutation ( k28c ) within dhfr . binding affinity of the k28c - dhfr 
 to tmp is similar to that of the wild type , indicating that the mutant 
 enzyme retains its affinity for tmp ; however , when the acrylamide - functionalized 
 ligand is used , a proximity - induced michael addition covalently links 
 the nucleophilic c28 residue on the mutant dhfr with the ligand . a 
 related development utilizing intramolecular tethering for covalent - dye 
 labeling makes use of fluorophore - binding peptides , called fluorettes 
 ( figure 10 ) . in this case , a reactive cysteine residue is 
 built into the linker region of the fusion protein construct made 
 up of a fluorette fused to the poi . the tethered small molecule is 
 designed with an -halocarbonyl function appended to a texasred - derived 
 fluorophore known to bind reversibly to the specific fluorette . the rapid noncovalent interaction between the 
 fluorophore and the fluorette templates a covalent reaction between 
 the -halocarbonyl motif on the tether and the proximal cysteine 
 on the linker attached to the target poi . covalent capture enables 
 stable dye incorporation through an acylamide 
 appendage that can conjugate with the engineered cysteine k28c - dhfr 
 mutant fused to poi . the crux of the method is that typically covalent interactions 
 occur slower than noncovalent associations . should a relatively rapid 
 noncovalent association facilitate covalent bond formation , the rate 
 enhancement relative to nonspecific covalent bond formation will allow 
 a specific covalent bond forming event to occur . thus , the best systems 
 will have low intrinsic reactivity of the reactive pole , or obviously , for this process to occur a nucleophilic residue ( usually 
 cysteine ) must be in close enough proximity to the ligand when bound 
 to its target to form a covalent bond to the acrylamide moiety . similar 
 considerations have been echoed by the pharmaceutical communities 
 as attempts are made to develop irreversible inhibitors from known 
 reversible binders . the acrylamide warhead demonstrated for the dhfr / tmp system is of 
 relatively low intrinsic reactivity / promiscuity , so its use will limit 
 off - target binding . it is thus ideally set to pair with trimethoprim , 
 which is a high - affinity , reversible , yet slow onset inhibitor of 
 dhfr . it is possible to increase the promiscuity of the reactive unit 
 appended to the ligand [ such as the -halocarbonyl in the fluorette 
 system ( figure 10 ) ] 
 to drive covalent attachment ; however , increasing reactivity will 
 typically elevate off - target binding , and will ultimately limit the 
 versatility of the method . clearly , such promiscuous reactive ligands 
 may not be kinetically compatible with slow onset inhibitor manifolds 
 either . similar ways to engender specificity have been achieved using 
 unnatural amino acids as in bolt for instance , and cyclooctyne such 
 as in proximity - enhanced aptamer ligation . one alternative solution 
 applicable to ligands with relatively long residence times on their 
 target is on - demand activation of a reactive functional group once 
 the recognition element has been bound to the poi and the excess washed 
 away . in this context , recently developed on - demand targeting or in situ unmasking ideas of proximity enhancement have appeared 
 in the literature ( vide infra ) . covalent capture concept 
 in stable dye incorporation using a fusion 
 peptide that bears a texas red fluorophore - binding sequence ( blue ) 
 and reactive cys residues . the pioneering lines of method development 
 within the class i framework over the recent decade have also highlighted 
 several general as well as specific challenges unique to individual 
 subclasses . both covalent and noncovalent modes of ligand binding are evident 
 in the examples illustrated above , but the decision as to which strategy 
 is better integrated in developing a new toolset is often not obvious . key aspects are the following : ( 1 ) reversible binders 
 can function catalytically allowing low ligand load while maintaining 
 high efficacy , whereas irreversible binders can not . ( 2 ) although typically assumed to be rapid , 
 binding equilibria of noncovalent ligands may be relatively 
 slowly established and occur in a complex , multistep process involving 
 enzyme conformational changes , especially for high - affinity binders . crystal structures of protein / ligand complexes will typically show 
 end - point conformations , and thus careful consideration of proximal 
 residues must be made especially if a class ib strategy is under consideration . ( 3 ) off - rates ( residence times ) of high - affinity reversible ligands 
 can also be prolonged , making washout of ligand prior to downstream 
 analysis , or sensing of analyte concentrations , require careful optimization . ( 4 ) covalent bond formation is typically slow ( detection 
 of a binding event may take minutes to hours ) , leading to time - dependent associations that need to be addressed 
 carefully to account for off - target effects and characterization of 
 ligand  protein interactions . ( 5 ) although not all off - target 
 interactions of irreversible target binders are necessarily covalent , 
 those that are will increase as a function of time . unlike for noncovalent off - target effects , the covalent off - targets , once formed , necessitate 
 new protein synthesis to regain activity if target inactivation has 
 occurred . alternatively , if gain of function has occurred , degradation 
 coupled with new protein synthesis is required . given the above , we 
 finally stress that conversion of noncovalent binders to covalent 
 binders in a bid to assess on- and/or off - target binding should be 
 approached with caution . one 
 trivial , but nonetheless common issue is that for classes ia and b , 
 care must be taken to ensure that tethered macromolecules forged by 
 the multifunctional small - molecule scaffolds ( either via intermolecular 
 protein  protein tethering enabled by the probes , or intramolecular 
 protein  ligand tethering ) are able to maintain functionality . an arduous series of chemical syntheses ( or permuted polypeptides ) , 
 optimizations of linkers and ordering of individual functional groups 
 along the tethered multifunctional small - molecule array are often 
 prerequisites of many successful class i methods discussed above . currently there are no clear predictive tools for achieving the optimal 
 design a priori . however , what has been less well 
 recognized is the fact that these considerations can also be used 
 to the researcher s advantage . in clash ( figure 6 ) , problems associated with steric repulsion have been turned on 
 their head by designing techniques based on steric - strain - directed 
 ligand binding and toggling . photo - bolt also utilizes rigidity - enhanced steric and stereoelectronic requirements 
 of intramolecular bond formation to its advantage ( figure 8) . one generally less attractive feature of systems 
 in which a bifunctional 
 ligand must interact with two independent binding sites is the prozone 
 or hook effect , wherein a drop in ternary 
 complex formation occurs at high ligand concentrations . this behavior 
 counteracts one of the key benefits of small molecules  their 
 predictable dose response . the hook effect occurs because once the 
 ligand is in excess relative to its target protein partners , there 
 is sufficient free ligand available for each of the two poles to bind 
 its respective protein target separately . although 
 this behavior has been helpful to model binding mechanisms in vitro , this complex operation confounds downstream biology , 
 especially in cells and whole organisms in which available small - molecule 
 ligand concentrations are not a simple parameter to control but often 
 dependent on pleiotropic factors . this problem is most applicable 
 to cases where both poles of the bifunctional probe interact reversibly 
 as in class ia systems discussed above . if one pole behaves as an 
 irreversible binder , the hook effect can be reduced , principally because 
 probe concentrations can be more readily controlled and excess can 
 be removed . on the other hand , the hook effect is less of an issue 
 if the ligand can behave catalytically as in next - generation protacs 
 because ligand concentrations can be minimized such that competition 
 for binding sites does not occur . finally , the hook effect may be 
 almost completely obviated in platforms such as bolt in which one 
 pole of the bifunctional probe reacts irreversibly and essentially 
 exclusively with its target site and the second binding site is also 
 on the same protein ( class ib systems ) . in terms of downstream applications , the majority of applications 
 using proximity - induced chemistry are limited by the complexity of 
 the molecules required to achieve their ultimate goal . thus , most 
 multicomponent tethered probes ( classes ia and ib)even those 
 currently at the cutting edge of this field  work well in cultured 
 cells , but much less information is available on how well they are 
 adaptable to applications in animal models or for human disease treatments . the molecular design inherently calls for 
 two poles that are each sufficiently complex to bind selectively to 
 a specific target poi , and a linker separating the poles . these prerequisites 
 render it challenging to create bifunctional small molecules that 
 adhere to basic drug design rules to optimize admet ( absorption , distribution , 
 metabolism , excretion , and toxicity ) properties , such as lipinski s rule of five . beyond the above considerations , increased size / complexity 
 of chimeric ligands may engender lower target specificity compared 
 to single - site binders . however , this proposition is not necessarily 
 true because the downstream application of single - site - binding ligands 
 and bifunctional molecules can be very different . single - site binders 
 often act as inhibitors of a specific enzyme , but dimerizers ( i.e. , 
 class ia ) induce gain of function through forcing protein  protein 
 associations . it is thus possible that by setting up low - occupancy 
 binding conditions ( i.e. , less than stoichiometric target - binding ) , 
 even chimeras derived from promiscuous ligands can have highly specific 
 effects . this is because formation of the desired protein complex 
 will cause a large gain in activity even at low ligand occupancy , 
 whereas unintentional off - target binding will be low occupancy and 
 is unlikely to usher a gain of function . the multicomponent scaffolding 
 is the centerpiece of both classes 
 ia and ib probe design in which each of the components is typically 
 derived from known ligands . unfortunately , not all protein ligands 
 are amenable to functionalization , either because they lack a suitable 
 functionalizable appendage , or because chemical modification perturbs 
 function . thus , there are relatively few bona fide ligand  protein pairs directly amenable to class furthermore , most of the functionalizable protein ligands are actually 
 inhibitors of a specific enzyme , principally because protein binders 
 have traditionally been identified from activity modulation screens . this caveat often limits downstream chemical biology applications 
 because protein targets are mostly restricted to those with an enzymatic 
 function , but that activity will be 
 inhibited as consequence of ligand binding . various methods have been 
 used to combat this deficiency , including the use of artificial domains 
 ( classes ia and ib ) such as dhfr . these considerations are not applicable 
 if a pole can be found that interacts with a site that is not functionally 
 coupled to the target enzyme s activity ; e.g. , the target protein 
 is part of a complex . for instance , in the case of protacs , catalytic 
 chemistry ( ubiquitination ) is carried out by the e2 ligase , which 
 is complexed to the target of the protac , an e3 ligase . more 
 recently , drug - screening methods that append macromolecular barcodes 
 onto small - molecule ligands have become widespread . these innovations provide a goldmine of potential small - molecule 
 ligands ( not necessarily inhibitors ) selective for a particular poi 
 ( not necessarily those with enzymatic functions ) that must be tolerable 
 to functionalization to appear as a hit . on the bright side , for proof 
 of principle , genetically encodable protein tags  such as snap , clip , dhfr , halo , and prime  can greatly extend the scope and generality of all 
 methodologies , provided the researcher is prepared to study ectopic 
 proteins and evaluate that the tag does not perturb protein function 
 drastically . the ease with which knockdowns / knockouts or inducible 
 systems can now be generated additionally provides 
 opportunities to study these fusion pois against the null background 
 or with minimal perturbation to the endogenous organizations . with 
 the advent of engineered immune cells for targeted therapy , there is also a distinct possibility that these artificial domains 
 could be used in  real - life  applications . the most 
 recent years have witnessed the emergence of proximity - directed 
 controlled release / delivery of reactive small - molecules in 
 situ . this method can obviate some of the problems associated with conventional 
 multicomponent tethering ( class i ) . instead of linking multiple small - molecule 
 modules through a chemical tether that remains unbroken throughout 
 the course of the assay ( figure 1 , classes ia and ib ) , a latent warhead is linked to 
 a recognition element for a specific poi . activation of the warhead 
 can be achieved ( a ) by sensitization ( generation of a reactive intermediate 
 upon contact with one pole of the ligand ) or ( b ) through photo - uncaging 
 of a protected , inert precursor of the reactive signal built into 
 one pole of the ligand , breaking the tether in the process ( figure 1 , class ii ) thereby 
 enabling proximity targeting to the poi . the basic design of class 
 ii  particularly the sensitization strategy  is not new ; 
 chromophore / fluorophore - assisted laser activation ( cali / fali ) methods 
 were introduced in 1988 . in these systems , reactive oxygen 
 species ( ros ) are overproduced in cells by excitation of photosensitizers  engineered 
 fluorescent proteins or dye molecules . poi - specific targeting is achieved 
 either via tethering organic chromophores / fluorophore  such 
 as malachite green or fluorescein  to poi - binding antibodies 
 ( delivered into cells via microinjection ) or small - molecule ligands . generality was subsequently shown through the use of tagging domains 
 fused to the poi . these early proof - of - concept studies required high 
 irradiation doses with focused laser beams and arc lamps that have 
 deleterious effects on cells . a further limitation 
 of the early method was off - target photodamage during irradiation , 
 stemming from the requirement for excess quantities of toxic dyes 
 that can not be easily washed away . thus , 
 high - affinity binding of the chromophore / fluorophore to the poi is 
 often a necessary factor . in this cali - based strategy , ru(ii)(tris - bipyridyl ) ( purple motif , inset ) functions as a photosensitizer for 
 the generation 
 of singlet oxygen ( o2 ) . binding of the peptoid 
 ( inset , blue motif ) to a specific poi ( e.g. , vegfr , 26s proteasome ) 
 allows targeted inactivation of poi by excess o2 produced by the ru catalyst upon irradiation with the visible light 
 of indicated parameters . to overcome some of these shortcomings , a more effective 
 visible - light - driven 
 singlet oxygen - generating cali reagent was recently developed by tethering 
 a [ ru(bpy)3 ] photocatalyst ( an 
 oxygen sensitizer ) to cell - permeable peptoids that are selective binders of pois ( figure 11 ) . ruthenium conjugate 
 inhibitors was demonstrated in direct comparison with their parent 
 compounds in light - triggered inactivation of the vascular endothelial 
 growth factor receptor 2 ( vegfr2 ) and 
 the chymotrypsin - like activity of the 26s proteasome ( figure 11 ) . in the 
 recent decades , many reports have indicated that endogenous electrophiles 
 can exert a specific redox - linked signaling role in cells . until recently , researchers used 
 bolus dosing with the reactive signal of choice to model these endogenous 
 signaling events . unfortunately , because of the promiscuity of these 
 reactive signals [ e.g. , > 400800 functional targets have 
 been 
 profiled for the reactive signaling molecule 4-hydroxynonenal ( hne ) ] , it is challenging to pinpoint gain - of - function 
 signaling responses that require only modest levels of modifications 
 on a single sensor protein . conditions of global stimulation and the associated off - target effects 
 can also lead to generation of secondary signaling metabolites / oxidative 
 stress that further confound analysis and data interpretation . motivated 
 to provide a gateway to track specific gain - of - function responses 
 brought about by non - enzyme - dependent redox - linked protein modifications , 
 our laboratory has pioneered the concept of targetable reactive electrophiles 
 and oxidants ( t - rex ) . a bifunctional small - molecule ligand was designed 
 with one pole 
 featuring the halotag - recognition unit , 
 a chloroalkane , and the other end bearing a photocaged precursor to 
 a specific reactive signal . photouncaging releases the signal with 
 temporal precision and in substoichiometric quantity with respect 
 to the target poi fused to halo ( figure 11 ) . the released entity can either react 
 with the target poi , or can diffuse away from the solvent cage , where 
 it is likely intercepted by reactive small - molecule thiols in the 
 cell such as glutathione . the engineered bacterial dehalogenase domain , 
 halotag ( 33 kda protein ) , ensures 
 target poi specificity , limits the amount of electrophile to be at 
 most stoichiometric to poi , and was shown to be inert to most reactive 
 electrophiles . t - rex has recently shown 
 that single - protein modifications by hne are sufficient to elicit 
 pathway activation . the commercial availability of the human and mouse 
 orfeomes as halo - tagged libraries also enables identification of novel 
 hne - sensing proteins using the halo - orfeome library in combination 
 with t - rex . cali / fali and t - rex embody the class ii proximity - directed 
 regimen and a comparison of these systems with classes ia and ib tethering 
 systems reveals interesting advantages and disadvantages . the key 
 advantage of the class ii is that the small molecule that would constitute 
 the second pole in class i is free to diffuse and is unaffected by 
 chemical modification . by contrast , class i requires the second pole  while 
 still tethered to the first pole  to be effective in poi binding . because of specific mechanistic differences , the class i approach 
 is compatible with irreversible and reversible ligands , whereas the 
 class ii approach only works for long residence ( most likely irreversible ) 
 binders . in contrast to class i in which high - selectivity ligands 
 are typically preferred , the aim of class ii is to endow a nonspecific 
 ligand with high specificity for a particular poi [ or for proteins 
 in a specific locale ( vide infra ) ] through proximity 
 enhancement . there is little to be gained from release of high selectivity 
 ligands unless its targeted delivery to one subcellular locale is 
 required . inert 
 photocaged precursors delivered to cells selectively and covalently 
 bind halotag fusion proteins ( inset ) and excess is washed away . photouncaging 
 driven by 365 nm light rapidly unleashes up to stoichiometric amounts 
 of reactive lipid - derived signaling electrophiles ( ldes ) from the 
 halotag as a point source ( inset , representative ldes ) . targeted lde 
 modifications on a specific poi in turn elicit gene - specific redox 
 signaling on demand in the backdrop of an unperturbed cell . copyright 
 2015 american chemical society . on the other hand , the differences between cali / fali and t - rex lie principally in their release stoichiometry . in 
 cali / fali , a sensitization event repeatedly allows activation of cellular 
 oxygen for the duration of the laser irradiation . in t - rex , a controlled 
 amount of a reactive entity to a maximum level of stoichiometry is 
 unleashed . thus , cali / fali protocols can generate a large quantity 
 of reactive signals ; t - rex , on the other hand , guarantees a substoichiometric 
 release of the reactive electrophile , mimicking substoichiometric 
 redox - linked modifications and signaling . thus , t - rex works best on 
 reactive sensor proteins , often rich in reactive cys that rapidly 
 capture highly reactive electrophiles : whereas , cali / fali can affect 
 proteins that are less reactive because reactive oxygen species can 
 be generated until photobleaching occurs . by similar logic , t - rex 
 is best used to study a small - molecule - initiated gain of function 
 ( or dominant loss of function ) or a new downstream signaling event , 
 rather than small - molecule - induced inhibition or loss of function . 
 because cali / fali is a targeted overload  method , it 
 works well to inhibit target proteins because 100% target protein 
 labeling is possible , although precision control of target poi labeling 
 stoichiometry may be more challenging . it is possible that spatial 
 positioning of the chromophore or caged 
 precursor , respectively , relative to the tethered protein will affect 
 targeted delivery in both cali / fali and t - rex . although this weakness 
 would be expected to apply most to t - rex , for the proof - of - concept 
 target poi , keap1 , the position of the halotag affects neither delivery 
 nor cys - residue specificity . this result is consistent with t - rex 
 targeting the most reactive cysteine(s ) in the target protein , which 
 are likely to be the ones that sense freely diffusive endogenous electrophilic 
 signals . finally , t - rex requires uv photo - uncaging ( figure 12 ) , which may limit utility 
 to cell culture and transparent organisms . some 
 methods closely related to cali / fali and t - rex have recently been 
 reported . these use proximity targeting through in situ unmasking of reactive entities with a goal of understanding interacting 
 proteins , or which proteins are present in a particular locale . biotin - dependent proximity - driven small - molecule chemistry has afforded 
 powerful insights into biological systems . this general strategy is 
 most readily exemplified by proximity - dependent biotin identification 
 also known as bioid . the heart of the 
 bioid platform is a reactive , adenosine monophosphate ( amp)-activated 
 biotin generated in situ . an engineered promiscuous 
 biotin ligase ( bira * ) is fused to the poi ( figure 13 ) . upon biotin stimulation , bira * creates 
 activated biotin that is subsequently released within the local environment 
 of the poi , enabling biotinylation of lysine residues within spatially 
 proximate ( 10 nm radius ) proteins . subsequent 
 streptavidin enrichment , peptidic digest , and ms proteomics permit 
 identification of unknown targets or validation of hypothesis - driven 
 binding partners to poi . a recent case of this innovative tool was 
 demonstrated with functional characterizations of large protein assemblies 
 in nuclear pore complex organization . bioid enables identification of proximate proteins for a 
 candidate poi fused to biotin ligase bira * upon stimulation of cells 
 with biotin . the practical labeling radius in cells is 10 
 nm . another biotin - dependent proximity 
 capture system has also emerged 
 aimed at ribosome profiling ( figure 14 ) . a spatially restricted biotin ligase ( bira ) fused to , for instance , 
 an endoplasmic reticulum ( er ) or mitochondrial - specific protein , is 
 coexpressed with ribosomes that are engineered to bear avitags , substrates 
 for bira . whole - cell stimulation with biotin causes proximity - targeted 
 biotinylation , enabling the elucidation of specific subgroups of ribosomes 
 at defined locations or with specific interacting factors . the method 
 was successfully applied to paint a detailed spatiotemporal picture 
 of local protein synthesis and posttranslational translocation at 
 the er as well as at er - associated ribosomes . this deep sequencing - based 
 method requires coexpression of spatially restricted ( e.g. , er or 
 mitochondrial membrane ) biotin ligase bira and ribosomes fused to 
 its substrate , avi tag . enrichment of biotinylated ribosomes 
 and subsequent deep sequencing of ribosome - protected mrnas inform 
 spatiotemporally precise message translation . in another innovation dubbed  apex  , an engineered 
 class i cytosolic pea or soybean ascorbate peroxidase ( 28-kda ) is 
 specifically targeted to a particular organelle using a localization 
 sequence temporal control is achieved 
 by 1 min h2o2 stimulation of the cells ( figure 15 ) . the nascent 
 short - lived radical thus formed covalently attacks electron - rich amino 
 acids on nearby protein targets . these endogenous biotinylated targets 
 are enriched with streptavidin beads , and identified by ms sequencing . this method has proven successful in answering key questions underlying 
 mitochondrial compartmentalization . apex . the ectopically expressed apex 
 peroxidase targeted to a specific 
 organelle ( in this case mitochondria ) catalyzes the oxidation of biotinphenol 
 by exogenously added h2o2 . the reactive biotinphenoxy 
 radical covalently labels proximate mitochondrial matrix proteins , 
 enabling proteomics profiling of biotinylatated proteins from defined 
 locations . the proximity - dependent biotin - derived capture 
 methods above have 
 proven powerful as discovery - based tools . the choice of capture method 
 used is dependent on many factors . since , in principle , any class 
 ii and related approaches can be used to profile associated or proximal 
 proteins , we catalog the key points to consider : ( 1 ) half - life of 
 the reactive molecule generated . this parameter defines the  radius 
 of influence  or distance over which a reactive small molecule 
 can  label  prior to decay . consensus second - order rate 
 constants for the reaction of probes with reduced glutathione , a biologically 
 relevant reference reacting partner , are shown in table 1 . this parameter will scale roughly 
 with bimolecular rate constants in table 1 , but is also a function of amount of probe 
 produced , and duration of experiment . this variable defines whether the signal can 
 be relayed to another protein post capture , thereby potentially diluting 
 the signal among secondary players . for instance , acyl phosphates , 
 like those generated during the course of bioid , can in principle 
 react with any nucleophilic residue including those bearing amines and thiols . the 
 latter creates a thioester , which can react with other nucleophilic 
 residues ( potentially on other proteins ) , whereas the former makes 
 a stable amide bond . acyl phosphates , 
 for instance , vary in their stability and reactivity from having second - order 
 kinetics ranging from slow ( undetectable ) to relatively fast as a 
 function of metal ion / lewis acid activation and ph of the microenvironment . radicals , 
 on the other hand , are intrinsically more reactive and of more transient 
 nature , making them more generally applicable . see references 
 for details . temperature not reported . at present , all biotin - dependent 
 capture methods rely on exogenous 
 small - molecule stimulation . in the case where the stimulant is a high 
 concentration of h2o2 such as in apex , even 
 though this is only for 1 min , it remains to be addressed how the 
 approach may affect the very proteins intended to be profiled . this 
 question is critical because exogenous h2o2 challenge 
 is known to perturb signal transduction pathways and organelle dynamics . for instance , 
 10-s pulsing with 1 m h2o2 reportedly 
 activates cellular redox relay chains and 5 min exposure to 1.5 mm h2o2 has been 
 shown to elicit ca - release channel activation . it is possible that a combination of a colocalized 
 point source of h2o2 ( generated on - demand in 
 a controlled manner ) and bira in conjunction with global biotin - phenol 
 administration could remedy this issue . using such a dual delivery 
 system , locale / protein specificity will likely be enhanced because 
 any aberrant apex or h2o2 generation would be 
 unlikely to colocalize . it should further be noted that apex and its 
 derivatives , as well as bira mutants , are the products of extensive 
 genetic engineering in which the desired activity is honed through 
 a combination of directed evolution , computational analysis , and rational 
 mutagenesis based on structural knowledge of the tagging enzyme . given the complexity of the systems 
 chemical biologists study , 
 there is no panacea to universally solve all problems . however , as 
 our discussion shows , there is a considerable amount of overlap between 
 all tethered processes , because they share common physiochemical principles . we thus recommend a thorough survey of tethered chemistry prior to 
 undertaking / designing experiments to try to anticipate problems , and 
 afford preemptive solutions . it is also paramount going forward that 
 we have as many tools available to study biology from a chemical perspective 
 as possible . for this reason , both class i and class ii approaches 
 should be considered when planning an experiment or developing a method . the tethered approach can endow low affinity / selectivity ligands / proteins 
 with laser - guided binding accuracy , and it can further force reactive 
 associations , which otherwise would require an unknown stimulus or 
 cue . it is thus often the case that the class i approaches require 
 that an outcome / association / end point be known or presumed ; for example , 
 a specific poi is inhibited , such as in bolt , or a specific poi forms 
 a tether through a specific sugar as in aptamer ligation , or a specific 
 poi is degraded through a specific pathway , such as with protacs . this requirement may be restrictive , however the power of small - molecule 
 tethering to promote an atypical cellular process will remain an important 
 arrow in the quiver of life scientists . this is principally because 
 gain of function is not a common mechanism for compounds that bind 
 single sites on enzymes , such as therapeutics . thus , there remains a huge scope to explore phenotypic output using 
 substoichiometric modification through gain - of - function - induced proximity 
 targeting . t - rex shows that on - demand targeting through provisional 
 or momentary 
 tethering can afford levels of specificity similar to those obtained 
 with permanent tethering . however , there is no evidence that t - rex 
 induces electrophilic modification of proteins that are not intrinsically 
 electrophile reactive , thus the target poi and reactive signal likely 
 should be chosen to be a  matched assuming that 
 the players are known , t - rex is another method to elicit gain - of - function 
 events in cells , and thus elicit  cellular mind control  . on - going experiments show that t - rex is a useful screening tool and 
 can identify the most reactive proteins in a panel of postulated sensor 
 proteins . beyond eliciting redox responses through on - demand redox 
 targeting , t - rex currently stands as a test case for future development 
 of synthetic systems that would enable precision - controlled gain - of - function 
 posttranslational modifications through , for instance , low - stoichiometry 
 acetylation and methylation events on a specific poi that in turn drive specific 
 biological outcomes . aside from cali , the other class ii - related approaches 
 are arguably best used to identify new interactions . this is because 
 class ii and related ideas are , by their nature , unbiased : only one 
 ligand pole is bound to a specific protein , leaving the other pole 
 free to react . it remains to be shown whether systems like t - rex 
 can be used in similar applications . a huge body of evidence 
 indicates that the proximity - dependent 
 approach has potential to be used in a wide array of circumstances . we anticipate future efforts will also be devoted to extending 
 these proof - of - concept design principles to testing in whole organisms 
 and ultimately in humans .
proximity 
 enhancement is a central chemical tenet underpinning 
 an exciting suite of small - molecule toolsets that have allowed us 
 to unravel many biological complexities . 
 the leitmotif of this opus 
 is  tetheringa strategy in which a multifunctional 
 small molecule serves as a template to bring proteins / biomolecules 
 together . 
 scaffolding approaches have been powerfully applied to control 
 diverse biological outcomes such as protein 
 protein association , 
 protein stability , activity , and improve imaging capabilities . 
 a new 
 twist on this strategy has recently appeared , in which the small - molecule 
 probe is engineered to unleash controlled amounts of reactive chemical 
 signals within the microenvironment of a target protein . 
 modification 
 of a specific target elicits a precisely timed and spatially controlled 
 gain - of - function ( or dominant loss - of - function ) signaling response . 
 presented herein is a unique personal outlook conceptualizing the 
 powerful proximity - enhanced chemical biology toolsets into two paradigms : 
  multifunctional scaffolding  versus  on - demand 
 targeting  . by addressing the latest advances and challenges 
 in 
 the established yet constantly evolving multifunctional scaffolding 
 strategies as well as in the emerging on - demand precision targeting 
 ( and related ) systems , this perspective is aimed at choosing when 
 it is best to employ each of the two strategies , with an emphasis 
 toward further promoting novel applications and discoveries stemming 
 from these innovative chemical biology platforms .