protein kinase ck2 
 is a pleiotropic , ubiquitous and intrinsically 
 active eukaryotic ser / thr protein kinase that is overexpressed in 
 various cancer types . in humans , ck2 forms 
 a heterotetrameric complex ( 2/2 ) consisting of two catalytic subunits ( ck2 or  ) attached 
 to a dimer of regulatory subunits ( ck2 or 2 ) .
the unique molecular architecture 
 of the ck2 holoenzyme could be exploited in the design of inhibitors 
 that do not target the atp site and thus provide a more specific mode 
 of action , prompting the discovery of various non - atp - competitive 
 inhibitors against ck2 . in particular , 
 significant efforts have been devoted to the development of compounds 
 that disrupt the transient , hetero - oligomeric protein  protein 
 interaction ( ppi ) between ck2 and ck2. given that the function of proteins critically depend on a correct 
 oligomerization state , and the importance of ck2 in modulating 
 the catalytic activity and substrate specificity of ck2 , disruption 
 of the constitutive , homodimeric ppi within ck2 represents 
 an alternative approach to interfere with ck2 function . developing small molecule inhibitors to disrupt ppis is a 
 challenging 
 task due to the typically extended and flat topology of contact surfaces , 
 often devoid of the well - defined deep clefts that are characteristic 
 of many enzyme active sites .
the difficulty is compounded 
 by the fact that the interacting surfaces of protein partners are 
 frequently segmented .
however , recent 
 successes in the development of ppi inhibitors have shown that ppis 
 are amenable to targeting by small molecules .
while the majority of the ppi inhibitors disrupt transient , hetero - oligomeric 
 ppis , only a comparatively few cases of small - molecule ppi inhibitors 
 that target constitutive , homo - oligomeric interfaces have been reported .
considering that achieving small - molecule inhibition of transient , 
 hetero - oligomeric ppis is already an inherently challenging effort , 
 the search for inhibitors that disrupt the constitutive oligomeric 
 interfaces within a protein is a potentially more difficult undertaking , 
 in part due to the typically higher affinity , larger interfaces , and 
 greater hydrophobic character of constitutive ppis .
interestingly , the hydrophobicity and the structural plasticity 
 of constitutive interfaces can enable small - molecule binding to form 
 structurally defined complexes .
thus 
 far , small - molecule oligomeric disruptors ( e.g. , spd304 , bio8898 , 
 6-hydroxydopa ) were discovered using a combinatorial library or high - throughput 
 library screen and targeted approach , with further studies revealing 
 their allosteric mode of inhibition .
only one recent study employed a fragment - based functional 
 screen to identify compounds whose inhibitory basis was disruption 
 of the dimeric architecture of a viral protease , rather than binding 
 to the active site .
in contrast 
 to traditional high - throughput screening , the use of 
 a smaller compound library in a fragment - based screen offers the advantage 
 of a more efficient and rapid exploration of weaker binding , but ligand - efficient 
 chemical moieties . here , a biophysical 
 fragment - screening cascade was performed to specifically identify 
 and validate fragments that are able to disrupt the ck2 dimer 
 interface .
this approach involved the sequential application of fluorescence - based 
 thermal shift to screen for preliminary hits , ligand - observed nmr 
 assays for validation of fragment binding , and native mass spectrometry 
 ( ms ) to confirm the ability of fragments to induce dimeric disruption . 
 an orthogonal biophysical assay using homodissociation isothermal 
 titration calorimetry ( itc )
activity 
 relationships ( sar ) governing dimerization affinity in a ck2 
 mutant and confirm the dimer - disrupting nature of the fragments .
the first 
 screening technique 
 is a fluorescence - based thermal shift ( fts ) assay , which monitors 
 protein thermal denaturation by using an extrinsic , environmentally 
 sensitive probe , for which the fluorescence increases upon binding 
 to the unfolded protein . as a protein is incrementally 
 heated , it unfolds and exposes its hydrophobic core
. unfolded protein 
 provides more nonpolar regions for protein - dye interaction , causing 
 a rise in the fluorescence intensity .
fragments that bind to and stabilize 
 or destabilize the protein will increase or lower the melting temperature 
 ( tm ) , respectively .
the difference between 
 the tm of the protein - fragment complex 
 and the tm of the apo protein represents the thermal shift ( tm ) .
as fluorescence - based thermal shift assay is the 
 first screening technique , its ability to inform whether small molecules 
 that induced the disruption of constitutive oligomeric interfaces 
 would produce thermal destabilization , corresponding to a loss of 
 stabilizing subunit interactions , when incubated with the protein 
 target was first evaluated ( figure 1a ) .
two eukaryotic macromolecular targets , rad52 and tnf- were selected for validation studies , 
 as small molecules that disrupt the constitutive oligomeric interfaces 
 in both proteins have been characterized .
binding of 6-hydroxydopa to rad52 induced 
 an undecameric - to - dimeric transition ( figure 1b ) .
no melting 
 curve could be observed for rad52 , indicating 
 that it has an extremely high thermal stability ( tm > 99.0 c ) , which is in agreement with published 
 data demonstrating the especially high melting temperature of a similar 
 rad52 construct , rad52 ( figure 1c ) . in the presence 
 of 6-hydroxydopa
, rad52 displayed an observable 
 melting transition , registering a tm of 
 85.0 c ( figure 1c ) .
spd304 , discovered from a combinatorial library screen , was observed 
 to eject a monomer of native trimeric tnf- by complexing with 
 a dimer of tnf- ( figure 1d ) .
similarly , the apparent tm of tnf- decreased from 65 c ( putatively 
 assigned due to the broad melt curve , which prevents precise tm determination ) to 61.0 c in the presence 
 of spd304 ( figure 1e ) .
the melting temperature ( tm ) of both 
 proteins decreased in the presence of the small - molecule oligomeric 
 disruptor , supporting the use of negative thermal shifts to identify 
 molecules that cause a dehomooligomeric transition .
use of fluorescence - based 
 thermal shift assay to detect small molecules 
 capable of inducing dehomooliogomerization . ( a ) hypothetical scheme 
 illustrating whether small - molecule ( triangle ) disruption of a homodimeric 
 assembly ( black ) to a monomeric state ( red ) is translated to a negative 
 thermal shift .
( b ) schematic showing the undecameric - to - dimeric transition of rad52 induced by 6-hydroxydopa ( green ) .
( c ) rad52 alone ( blue ) did not produce a melting transition , 
 indicating that its tm was more than 99 
 c . in the presence of 6-hydroxydopa , rad52 registered a tm of 85.0 c ( red ) . 

( d ) spd304 ( blue ) causes the dissociation of native trimeric tnf- 
 ( purple ) into a spd304bound dimer ( pink ) and monomer ( yellow ) . 

( e ) the melting curve of native trimeric tnf- ( purple ) showed 
 that it had a putative tm of 65 c . 
 in the presence of spd304 ,
( f ) distribution of thermal shift 
 values induced by fragments in ck2 from the fluorescence - based 
 thermal shift screen . in light of these results , 800 fragments were screened at 
 5 mm 
 against ck2. a histogram depicting the distribution of tm induced by the fragments is shown in figure 1f .
most of the fragments induced ck2 destabilization as shown 
 by the left - skewed distribution of tm values .
the maximum stabilizing and destabilizing tm from the screen was + 0.8 c and 6.0 
 c , respectively .
while no fragments induced significant positive 
 thermal shifts , it was interesting to note that several fragments 
 significantly lowered the melting temperature of ck2. fragments 
 which induced a tm < 1.5 
 c for the negatively shifting fragments were selected for follow - up 
 studies .
ligands that increase 
 the tm of a protein 
 are predominantly pursued for subsequent validation and optimization , 
 as they cause stabilization of the protein  ligand complex .
in contrast , it is commonly believed that fragments that cause negative 
 thermal shifts signify preferential fragment binding to the unfolded 
 form of the protein , and are subsequently excluded from further analysis . however , this does not necessarily hold true 
 for all protein systems , as seen in the validation studies using rad52 and tnf- and their disruptors . to our knowledge , 

only one fragment - based study selected both thermally stabilizing 
 and destabilizing fragments against homodimeric mycobacterium 
 tuberculosis bioa ( an aminotransferase that uses a pyridoxal 
 5-phosphate cofactor ) for follow - up studies , although no rationale 
 was given for considering thermally destabilizing fragments .
only one out of the 12 destabilizing fragments from the original 
 screen against bioa produced a structure by cocrystallization .
subsequent sar - by - catalog of fragment hits resulted 
 in the crystallographic and thermodynamic characterization of a series 
 of inhibitors .
this study has shown that thermally 
 destabilizing fragments can be inhibitors and that caution should 
 be applied before rejecting negatively shifting fragments for further 
 evaluation .
furthermore , it has been suggested that thermally destabilizing 
 fragments may have an additional value in promoting a more rapid degradation 
 of the target protein .
both spd304 
 and 6-hydroxydopa , which promoted subunit disassembly 
 by binding to a non - native form of their protein target , lowered the 
 melting temperature of their protein complexes .
the disruption of 
 stabilizing subunit interactions between protomers in an oligomeric 
 protein by any ligands could be expected to decrease the protein s 
 stability , which would be reflected by a lowering of its melting temperature . 

essentially , any interpretation of fts results must take into consideration 
 that the fts assay depends on fluorescent dye binding .
changes in 
 the tm are a reflection of changes in 
 the fluorescent dye binding .
a ligand that binds to and stabilizes 
 a protein would slow both its denaturation and exposure of its hydrophobic 
 region , causing a delayed rise in the fluorescence intensity to result 
 in an increase of the tm . on the contrary , 
 a ligand that disrupts constitutive hydrophobic interfaces in an oligomeric 
 protein would allow the fluorescent dye access to hydrophobic environments 
 for binding , leading to an earlier increase in the fluorescence intensity 
 to produce a decrease in the tm .
structural 
 analyses of both rad52 and tnf- revealed 
 that oligomerization is largely mediated by hydrophobic interactions 
 between the subunits . by causing earlier and greater 
 exposure of hydrophobic areas upon subunit dissociation , spd304 and 
 6-hydroxydopa stabilized non - native forms of their protein complex 
 that bind the dye with higher propensity than the native form , thereby 
 resulting in an early rise in the fluorescence intensity to eventuate 
 in negative thermal shifts . on the basis of this reasoning , coupled 
 with the hydrophobically driven nature of ck2 dimerization , 
 a possible model that fits our results is that thermally destabilizing 
 fragments are causing monomerization of ck2. a model to describe fragment - induced negative thermal shift 
 of ck2 is presented in figure 2 .
alternatively , there is also a possibility that a 
 small proportion of ck2 monomer could be present that provides 
 a rapid route for unfolding .
furthermore , in the vein of a mechanism 
 resembling the effect of bio8898 on cd40 , thermally destabilizing 
 fragments could also bind to and distort the ck2 dimer interface 
 without completely causing subunit dissociation . in the absence of additional experimental evidence , it 
 is difficult to speculate whether dimer distortion translates to a 
 negative thermal shift .
however , subsequent orthogonal assays using 
 native ms and homodissociation itc would help clarify the most probable 
 molecular mechanism . model of fragment binding to ck2 ( 2 ) to 
 rationalize negative thermal shift .
fragment - induced monomerization 
 of 2 results in the formation of a species ( denoted 
 with an asterisk ) with a higher hydrophobic character that promotes 
 earlier binding of the fluorescent dye , thereby resulting in negative 
 thermal shift .
the thermal shift screen can be 
 applied to proteins that experience 
 either reversible or irreversible denaturation . a reversible two - state 
 equilibrium between the structured native state and the unfolded state 
 can be used to describe protein unfolding , with the assumption that 
 only these two states exist . on a sufficiently short time scale ,
it 
 has been experimentally and computationally shown that the unfolding 
 process is pseudoreversible , as it is possible to generate reasonable 
 plots of the apparently irreversible denaturation process using the 
 vant hoff equation , which relates changes in the equilibrium 
 constant to temperature . over the entire 
 time course of a thermal shift experiment
, the exposure of hydrophobic 
 cores during protein unfolding would ultimately lead to the formation 
 of irreversible aggregates .
therefore , a majority of large multidomain 
 proteins will eventually undergo partially or completely irreversible 
 denaturation . using proteins that undergo 
 irreversible thermal protein denaturation , such as e.  coli aspartate transcarbamoylase and the core protein of the lac repressor
, 
 it has been shown that the denaturation process obeys equilibrium 
 thermodynamics as characterized by the vant hoff equation , 
 thus resembling a reversible process .
furthermore , 
 the data obtained from simulating an irreversible denaturation process 
 were similar to that of a completely reversible denaturation model .
therefore , thermal shift screening could be 
 applied to oligomeric proteins regardless whether they undergo reversible 
 or irreversible denaturation .
as fragments enable sampling of 
 a larger chemical space , fragment 
 libraries tend to be smaller than a high - throughput screen library .
thermal shift screening is rarely rate limiting , 
 but could be achieved more rapidly with the use of a higher temperature 
 ramp rate . in general , the magnitude of thermal shift is not strongly 
 dependent on the temperature ramp rate when a heating rate between 
 18 c min is applied .
this is supported 
 by a study in which thermal shifts produced by screening different 
 concentrations of known ligands against nine proteins did not change 
 significantly despite the use of ramp rates spanning 18 c 
 min .
it was further 
 recommended that a heating rate of up to 4 c min could be used with minimal impact on ligand detection for most proteins .
two nmr approaches could be employed to 
 validate fragment binding 
 to the protein target : ligand - observed and protein - observed nmr .
ligand - observed 
 nmr assays are more popular as there is no requirement to produce 
 isotopically labeled proteins . as there is no upper limit on the protein 
 size , ligand - observed methods
furthermore , the assays are 
 straightforward , rapid , and have relatively low protein consumption 
 by enabling the acquisition of multiple nmr assays on the same sample . 

the principles and applications of ligand - based nmr methods have been 
 extensively reviewed .
briefly , ligand - observed nmr 
 assays depend on monitoring differences in the properties of the ligand 
 spectra ( e.g. , magnetization transfer or relaxation ) upon interaction 
 with the macromolecular target .
experiments based on direct or indirect 
 magnetization transfer ( saturation transfer difference [ std ] and water  ligand 
 observed via gradient spectroscopy [ waterlogsy ] ) and differential 
 relaxation ( carr  purcell  meiboom  gill [ cpmg ] ) 
 are commonly used in fragment - based campaigns .
performing three orthogonal nmr 
 assays , each with their own advantages and disadvantages , lowers the 
 chances of false positives and negatives originating from artifacts 
 of a single nmr experiment .
all the 60 destabilizing fragment 
 hits identified from the thermal shift screen were validated for binding 
 to ck2 using a panel of three ligand - observed h 
 nmr assays ( std , waterlogsy and cpmg ) .
the nmr screen validated 45 
 of the 60 destabilizing hits , representing a 75% validation rate , 
 thus giving a good confidence of the binding event ( supplementary figure s1 ) . among the destabilizing hits , 40 
 fragments showed binding in all three nmr experiments , a further 5 
 fragments showed binding in at least two nmr experiments ( supplementary figure s1 ) .
no correlation between 
 the degree of binding observed in the nmr experiments and the magnitude 
 of tm values was observed ( supplementary table s1 ) .
the magnitude of thermal 
 shift is a combined function of the enthalpy change of protein unfolding , 
 enthalpy change of ligand binding and ligand affinity .
the magnitude of thermal shift of a set of compounds 
 will correlate to their binding affinities only when the compounds 
 possess similar binding enthalpies .
this has been demonstrated by 
 a study in which the rank order of affinity and binding constants 
 of a series of chemically and structurally distinct -site amyloid 
 precursor protein - cleaving enzyme 1 ( bace1 ) inhibitors obtained using 
 fts and itc were found to correlate well , particularly because the 
 ligands had similar binding enthalpies .
had the binding enthalpies of the bace1 inhibitors been very different , 
 the affinity ranking based on the thermal shift values would be inaccurate . 

this implies that the tm value 
 associated with fragment binding does not necessarily correlate with 
 its binding affinity .
as the ck2 fragment hits are chemically 
 and structurally different , it is possible that they have different 
 binding enthalpies .
this means that fragments with the same binding 
 affinity , but with different binding enthalpies will generate different 
 tm values .
hence , the extent of 
 thermal destabilization can not be used as a measure of its binding 
 affinity , and , by extension , its degree of binding in nmr assays .
mass 
 spectrometry ( nanoesi  ms ) provides rapid , sensitive , label - free 
 and accurate detection of noncovalent assemblies , such as protein 
 oligomers or protein  ligand complexes , in the gas phase .
various studies have shown that gas - phase proteins 
 retain folded conformations and possess structural features that approximate 
 to those in the solution state , thus providing a simulacrum of solution - phase 
 conditions .
the high separation efficiency 
 of ms is especially relevant for examination of the oligomeric populations 
 of proteins in the gas phase . for similar 
 protein species
, it has been shown that there is good agreement of 
 the oligomeric distribution obtained using gas - phase and solution - phase 
 methods , although the caveat that similar oligomeric forms may have 
 different efficiency of ionization , transmission and detection must 
 be recognized . nevertheless , adopting a native ms approach enables us to address 
 the presence and degree of oligomeric state perturbation by thermally 
 destabilizing fragments , which could serve as an indication of the 
 dimer - disrupting potency of fragments .
native ms was used to 
 study the effect on the oligomerization state of ck2 of the 
 40 destabilizing fragment hits that were shown to bind to ck2 
 in all three ligand - observed nmr assays .
native mass spectra of 16 
 m ck2 in the presence of 5% ( v / v ) dmso were acquired 
 under non - denaturing conditions by nanoesi 
ck2 produced 
 two well - resolved narrow charge state distributions corresponding 
 to monomeric ck2 ( observed mass = 22  962  17 da ; 
 calculated mass = 22  945 da ) and dimeric ck2 ( observed 
 mass = 45  938  17 da ; calculated mass = 45  890 
 da ) , with lowly charged ions to signify that they retain folded , native - like 
 structures ( figure 3 ) .
the predominant species is dimeric ck2 , which is consistent 
 with published structural data .
native mass spectra of 
 16 m ck2 , acquired in 0.5 
 m ammonium acetate ph 8.0 , in the presence of validated thermally 
 destabilizing fragments ( 2 mm ) .
the percentage of ck2 monomerization 
 induced by a fragment is indicated in orange text below the compound 
 number .
the 
 observed mass and identity of each species are indicated beside the 
 symbols .
only one charge state of each species is indicated in the 
 spectra . in the presence of 2 mm fragment
, 
 native ms showed that 18 out 
 of the 40 compounds induced a higher population of monomeric ck2 
 by promoting the disassembly of dimeric ck2 to different extents 
 ( 1871% monomerization ) ( figure 3 ) .
1 and 2 had the greatest 
 effects on dimer disruption , inducing 71% and 67% monomerization of 
 ck2 , respectively .
both 1 and 2 possess 
 the 5-substituted pyrazole core , suggesting the importance of this 
 chemical scaffold in mediating dimer disruption .
furthermore , native 
 ms experiments showed 3 and 4 , bearing the 
 pyrazole scaffold , to cause 49% and 24% monomerization of ck2 , 
 respectively .
apart from pyrazole - based fragments , compounds with 
 quinoline ( 57 ) and naphthol ( 811 ) cores were also well represented 
 ( supplementary figure s2 ) .
there was no 
 correlation between extent of monomerization and thermal shift , as 
 the magnitude of thermal shift induced by a fragment is not necessarily 
 proportional to its affinity for the protein .
ablation of the 
 zinc finger , by mutation of the zinc - coordinating cysteine residues , 
 resulted in dimerization - defective ck2. the observed mass of monomeric ck2 ( 22  962 
 da ) is in close agreement with the theoretical mass of monomeric ck2 
 with one zn bound ( 22  945 da ) , showing that fragments 
 do not cause monomerization by metal sequestration .
importantly , the 
 narrow charge state distribution observed for monomeric ck2 
 signifies that the destabilizing fragments were able to cause dimeric 
 disruption of ck2 without denaturing the protein .
furthermore , 
 the clear observation of an enrichment in the monomeric species shows 
 that ck2 is converted to the monomer at the fragment concentration 
 used .
this argues against the possibility that fragments were merely 
 distorting the ck2 dimer interface without inducing dissociation 
 ( reminiscent of the effect of bio8898 on cd40 ) , as an increase in 
 the intensity of the monomeric species would not be expected .
however , it is possible that the fragments could 
 be both distorting and weakening the ck2 dimer interface to 
 the extent of causing dissociation .
information about binding 
 stoichiometry could not be extracted 
 from the native mass spectra , as only non - complexed monomeric and 
 dimeric ck2 were detectable .
this is not unusual given that 
 the fragments may be potentially mediating the disruption of dimeric 
 ck2 by mainly engaging in hydrophobic interactions with residues 
 at the dimer interface .
the hydrophobic effect does not apply for 
 proteins in the gaseous phase , and protein  ligand complexes 
 bound primarily by hydrophobic interactions tend to dissociate in 
 the gas phase .
four highly conserved cysteine 
 residues ( cys109 , cys114 , cys137 and 
 cys140 ) in a ck2 protomer are involved in zinc coordination 
 to form a zinc - binding motif that constitutes the dimerization domain .
the dimerization of ck2 is largely driven 
 by hydrophobic interactions , with the / core composed 
 of nonpolar residues ( pro110 , val112 , leu124 , val143 , and the hydrophobic 
 moieties of tyr113 and tyr144 ) ( supplementary figure s3 ) .
salt - bridge and hydrogen - bonding interactions ( arg111 
 and asp142 , backbone carbonyl and amino groups of pro110 and thr145 , 
 and val143 and val112 , respectively ) also serve to stabilize the dimer 
 ( supplementary figure s3 ) .
dimerization 
 of ck2 results in the burial of 1766  per 
 protomer , a value consistent with that expected of a permanent ppi , 
 establishing ck2 as an obligate dimer .
a homodissociation itc assay was 
 developed in order to provide an orthogonal , solution - phase approach 
 of confirming the mechanism of fragment - induced dimer - disruption and 
 examine structure  activity relationships governing dimerization 
 affinity . in homodissociation itc , a concentrated solution of oligomer 
 is titrated into a buffer cell using a series of small - volume injections .
the initial few injections lead to huge dilutions 
 of the protein concentration , and therefore promote oligomeric dissociation . 

each injection typically yields an endothermic heat pulse , which progressively 
 decreases in intensity over the entire course of titration due to 
 an increase in the protein concentration in the cell disfavoring dissociation . 

the oligomer dissociation constant is determined by fitting the data 
 to a dissociation model , operating with the assumption that the monomer 
dimer 
 equilibrium is reversible under the experimental conditions . given the weak affinity expected for fragment binding , a strategy 
 of directed mutagenesis was adopted to systematically reduce the strength 
 of the dimeric ck2 interface ( supplementary figure s3 and supplementary figure s4 ) .
being core hydrophobic 
 residues that significantly contribute to the stabilization of the 
 dimer interface , pro110 and val143 were mutated to aspartate to attenuate 
 hydrophobic interactions and introduce electrostatic repulsion to 
 weaken subunit association .
/ v143d 
 was shown to exist in a monomer  dimer equilibrium with a kd of 90 m in the presence of the vehicle 
 control , dmso ( figure 4a ) .
thermally destabilizing fragments decreased the dimerization affinity 
 in ck2 p110d / v143d in homodissociation itc experiments . ( a ) 
 homodissociation isotherm of ck2 p110d / v143d in 5% ( v / v ) dmso . 

( b ) homodissociation isotherms of 4 and its analogues 
 ( 4a and 4b ) . ( c )
the top and bottom panels of each itc profile illustrate 
 the raw calorimetric data and the integrated heats per injection , 
 respectively . generally , all the 18 
 fragments that induced monomerization of 
 ck2 dimer in native ms experiments decreased the dimerization 
 affinity of the double mutant , suggesting that they disrupted the 
 dimeric interaction in ck2 p110d / v143d ( supplementary table s2 ) .
this was also supported by the appearance 
 of the dissociation isotherms , wherein the intensities of the endothermic 
 heat pulses increased and the dilution isotherms became more attenuated 
 in the presence of fragments , indicating greater dimer dissociation 
 ( supplementary figure s5 ) .
there is no 
 correlation between the magnitude of thermal shift , or the degree 
 of monomerization obtained from native ms experiments and the dimerization 
 affinity ( supplementary table s1 ) .
this 
 could be attributed to the use of a mutant construct with different 
 interfacial properties from that of wild - type ck2. out of all 
 the 18 fragments tested , 2 was the most potent in mediating 
 dimerization disruption ( kd = 1010 m ) . 
 surprisingly , 1 only caused a modest weakening of dimerization 
 affinity ( kd = 200 m ) , despite 
 inducing the greatest extent of monomerization in the native ms assay . 

this suggests that the binding site of 1 could have been 
 affected by the double mutation , and that 2 might be 
 binding to a different region of ck2 from 1 .
further screening of structural 
 analogues of 4 and 16 , available from our 
 in - house compound collection , resulted in the identification of more 
 potent dimer - disrupting compounds .
the effects of functional group 
 substitutions on 4 ( kd = 
 460 m ) ( figure 4b ) and 16 ( kd = 230 m ) 
 ( figure 4c ) were explored , 
 with the kd values reflecting the apparent 
 affinity for dimer formation , and not the affinity of compound binding . changing the chloro group in 4 to a hydroxyl group 
 preserved a similar dimer - disrupting potency ( 4a , kd = 410 m ) ( figure 4b ) . by combining the observation
that 4a was able to hinder dimerization of the double mutant with 
 the fact that 1 demonstrated the greatest monomerizing 
 effect in native ms , we examined whether 4b , with the 
 3-bromo and 5-phenyl groups in 1 replaced with phenolic 
 groups , would have a greater potency toward effecting dimeric disruption . 

indeed , 4b caused a significant decrease in the dimerization 
 affinity ( kd = 1,200 m ) ( figure 4b ) , suggesting that 
 polar interactions and hydrophobic or aromatic stacking interactions 
 contribute to weakening dimeric association in the ck2 double 
 mutant . replacing the methylene linker in 16 with 
 an nh group 
 caused approximately 2fold increase in dimer - disrupting effect 
 ( 16a , kd = 490 m ) , 
 suggesting a role for hydrogen bonding interactions in effecting subunit 
 disassembly ( figure 4c ) .
incorporation of functional groups at different positions on 
 the phenyl ring , however , had different effects .
addition of an ester 
 group at the para position of the phenyl ring resulted 
 in a decrease in dimer - disrupting potency ( 16b , kd = 230 m ) .
substitutions at the meta position of the phenyl ring of 16a were 
 generally more favorable for dimer disruption than para substitutions as shown by the lower dimerization affinity induced 
 by 16c ( kd = 690 m ) 
 and 16d ( kd = 350 m ) 
 than 16b ( figure 4c ) .
the sar studies have demonstrated that the ck2 
 p110d / v143d 
 mutant could potentially serve as a surrogate protein for the development 
 of fragments into more potent compounds that disrupt the ck2 
 interface .
in addition , the other ck2 mutant proteins provide 
 a range of weaker homodimeric interfaces ( i.e. , ck2 p110d / r111d , 

ck2 p110d / v112d / v143d ) ( supplementary figure s3c ) that could be useful for the systematic screening and 
 development of compounds that destabilize the homodimeric interface 
 of wild - type ck2. having established that thermally destabilizing fragments drive 
 the 
 dimeric - to - monomeric transition of ck2 , what could be the potential 
 consequences of such an effect ? despite being able to interact with 
 ck2 to form the heterotetramer , the ck2 p110d / v143d 
 mutant decreased the catalytic activity of ck2 , highlighting 
 that the modulation of ck2 catalytic activity by ck2 
 is highly dependent on a proper dimeric architecture of ck2. cell studies have shown that a dimerization - incompetent 
 ck2 , generated by mutating two conserved cysteine residues 
 of the zinc finger to serine , was defective in forming the 2/2 heterotetramer and experienced faster 
 degradation .
together , these studies 
 suggest that dimer - disrupting fragments could promote ck2 degradation 
 and an attenuation of ck2 catalytic activity through favoring 
 the formation of the ck2 monomer .
in 
 summary , this study demonstrated the application of a fragment - based 
 approach to specifically identify small molecules with the ability 
 to induce disruption of the ck2 dimer .
orthogonal biophysical 
 experiments involving native ms and homodissociation itc support a 
 mechanism that is consistent with fragment - induced dimeric disruption . 

future work in obtaining cocrystal structures of ck2 with the 
 destabilizing fragments would help to elucidate the structural determinants 
 of dimeric disruption and enable structure - guided optimization of 
 compounds .
the approach described in this study could potentially 
 be applied to discover small molecules to disrupt other therapeutically 
 relevant and challenging homo - oligomeric proteins as a means of modulating 
 protein function .
bacterial 
 expression vectors encoding sequences for homo sapiens ck2 and ck2 mutants ( all encoded within pgex-6p-1 ) were transformed into e.  coli bl21(de3 ) .
a freshly transformed colony was 
 inoculated into lb broth supplemented with ampicillin and grown overnight 
 at 37 c . after inoculation of overnight culture , lb cultures 
 were grown at 37 c , induced with 0.3 mm iptg after reaching 
 an optical density of 0.6 (  = 600 nm ) and allowed overnight 
 expression at 18 c .
harvested cell pellets were suspended and 
 sonicated in cold lysis buffer a ( 50 mm tris 
cellular debris was removed 
 by centrifugation ( 20  000 rpm , 30 min , 4 c ) and ck2 
 was purified using glutathione sepharose 4b beads ( ge healthcare ) . 

the beads were washed with 20 column volumes of cold buffer a and 
 incubated with 3c protease at 4 c overnight to cleave the gst 
 tag .
the digested protein solution was loaded onto a hitrap q column 
 ( ge healthcare ) and fractionated over a 01000 mm nacl gradient 
 buffered with 50 mm tris 
hcl ph 8.5 and 2 mm -mercaptoethanol . 
 ck2-containing fractions , analyzed by sds 
page , were 
 concentrated and loaded onto a superdex 200 26/60 column ( ge healthcare ) 
 equilibrated with cold buffer b ( 50 mm tris 
mutagenesis of homo 
 sapiens ck2 to generate the 
 p110d , p110d / r111d , p110d / v143d and p110d / v112d / v143d mutants was 
 performed using the q5 site - directed mutagenesis kit ( new england 
 biolabs ) according to the instruction manual .
vectors of mutant clones 
 were sequenced ( dna sequencing facility , university of cambridge ) 
 to verify correct incorporation of mutation .
the expression vector encoding homo sapiens rad52 ( cloned into pet28 ) 
 was transformed into e.  coli bl21-codonplus(de3)-ripl . 

fresh transformants 
 were inoculated into lb broth supplemented with kanamycin and chloramphenicol , 
 and grown overnight at 37 c .
after inoculation of overnight 
 culture , lb cultures were grown at 37 c , induced with 1 mm iptg 
 after reaching an optical density of 0.6 (  = 600 nm ) and allowed 
 expression at 30 c for 4 h. cell pellets were suspended and 
 sonicated in buffer a ( 50 mm tris 
debris was removed by centrifugation ( 20  000 rpm , 30 min , 4 
 c ) and rad52 was purified using ni 
the beads were washed with buffer a supplemented 
 with 20 mm imidazole , and eluted with buffer a supplemented with 300 
 mm imidazole .
the eluted protein solution was concentrated and loaded 
 onto a superdex 200 26/60 column ( ge healthcare ) equilibrated with 
 buffer b ( 50 mm tris 
fractions containing rad52 were combined and 
 loaded onto a hitrap heparin hp ( ge healthcare ) and washed with buffer 
 b. rad52 was eluted using a 2001000 
 mm kcl gradient over 20 column volumes .
all proteins 
 produced in - house 
 were assessed for their identity , purity , monodispersity and oligomeric 
 state using a combination of sds  page , amino acid analysis , 
 dynamic light scattering and native mass spectrometry ( supplementary figure s6 ) .
the thermal shift 
 assay was performed on a lightcycler 480 real - time pcr system ( roche ) 
 in 96-well white plates ( roche ) . for rad52 , 
 each well contained 40 l of 2 m rad52 and 2.5 sypro orange in 50 mm tris 
hcl ph 7.5 , 200 
 mm kcl , with 6-hydroxydopa ( santa cruz biotechnology ) added to a final 
 concentration of 2.5 mm in 5% ( v / v ) dmso .
for tnf- ( gibco ) , 
 each well contained 40 l of 10 m tnf- and 5 
 sypro orange in 50 mm tris 
hcl ph 8.5 , 200 mm nacl , with spd304 
 ( cambridge bioscience ) added to a final concentration of 200 m 
 in 5% ( v / v ) dmso . for ck2 ,
each well contained 40 l 
 of 6 m ck2 and 5 sypro orange in 50 mm tris 
fragments were tested at a final concentration 
 of 5 mm in 5% ( v / v ) dmso .
each plate was sealed with an optically 
 clear foil and centrifuged for 1 min at 1000 rpm before performing 
 the assay .
the fluorescence intensity was 
 measured with ex = 480 nm and em = 580 nm .
the melting temperature ( tm ) was obtained by determining the minimum of the first derivative 
 curve of the melt curve . the thermal shift ( tm )
was determined by computing the difference between 
 the tm of the protein in the presence 
 of compound and that of the protein in the presence of 5% ( v / v ) dmso .
ligand - observed h nmr experiments were performed at 278 k on a 700 mhz bruker 
 nmr spectrometer fitted with a 5 mm triple txi cryoprobe .
samples were 
 made up to 200 l in 3 mm capillaries with trimethylsilylpropionic 
 acid - d4 ( tsp ) for calibration .
negative 
 control ( no protein ) experiments were performed for each compound 
 tested .
all binding experiments were carried out using 20 m 
 ck2 in 50 mm tris 
hcl ph 8.5 , 50 mm nacl , 20 m 
 tsp , 10% ( v / v ) d2o and 0.01% ( v / v ) tween20 .
fragments 
 were tested at 2 mm in a final concentration of 24% ( v / v ) 
 dmso - d6 in binding experiments .
spectra were recorded on a 
 synapt hd mass spectrometer ( waters ) modified for studying high masses . 

ck2 was exchanged into 0.5 m ammonium acetate solution ph 8.0 
 using micro bio - spin 6 chromatography columns ( bio - rad ) .
two mm of 
 a fragment was incubated with 16 m ck2 for 30 min before 
 analysis .
2.5 l 
 of protein solution was electrosprayed from a borosilicate emitter 
 ( thermo scientific ) .
typical conditions were capillary voltage 1.61.8 
 kv , cone voltage 6080 v , collision voltage 1020 v , 
 with backing pressure 34 mbar and source temperature of 20 
 c .
the concentration of ck2 p110d / v143d was selected 
 such that the heats of dissociation afforded a good signal window 
 and that baseline is reach in the presence of the vehicle control , 
 indicating no further dissociation .
the syringe solution consisted 
 of 600 m ck2 p110d / v143d incubated with 5 mm fragment 
 in 50 mm tris 
the cell solution 
 consisted of 50 mm tris  hcl ph 8.5 , 50500 mm nacl . 
 both the syringe and cell solutions contained dmso at a final concentration 
 of 58% ( v / v ) .
the titration consisted of 19 injections of 
 2 l of the syringe solution every 120 s. each fragment  protein 
 mixture was subjected to a single titration .
errors for quoted kd values represent errors of the curve fit from 
 a single experiment .
data were fitted and analyzed using the dissociation 
 model in the microcal peaq  itc software ( malvern ) .
 identifying small molecules that 
 induce the disruption of constitutive 
 protein  protein interfaces is a challenging objective . here , 
 a targeted biophysical screening cascade was employed to specifically 
 identify small molecules that could disrupt the constitutive , homodimeric 
 protein  protein interface within ck2. this approach 
 could potentially be applied to achieve subunit disassembly of other 
 homo - oligomeric proteins as a means of modulating protein function . 
Introduction
Results
and Discussion
Conclusion
Methods