over the last decade , fragment - based lead discovery ( fbld ) has been adopted throughout academia and industry as a tool to identify viable starting points from which lead compounds can be generated . to date , at least nine fbld projects have led to compounds in phase ii or iii studies , including one approved drug .
the fbld approach aims to identify very small molecular fragments ( 150300 da ) that bind to specific sites of target proteins and are subsequently grown , merged , or linked to produce more potent drug leads .
a variety of biophysical techniques have been applied in fbld campaigns , ranging from protein and ligand - observed nuclear magnetic resonance ( nmr)based approaches , x - ray crystallography , surface plasmon resonance ( spr ) , and mass spectrometry ( ms ) to isothermal titration calorimetry ( itc ) and thermal shift assays .
each method has advantages and disadvantages in terms of sample consumption , degree of automation , and assay complexity . to fully exploit the benefits of fbld
, these biophysical methods should be fast , efficient , and precise in their ability to characterize binding fragments , while also reducing the number of false positives and false negatives .
microscale thermophoresis ( mst ) is an emerging technique that has been broadly applied to investigate biomolecular interaction of a variety of drug targets .
mst detects the directed movement of fluorescent molecules in microscopic temperature gradients in microliter - volume capillaries to quantify interaction affinities ( suppl .
each molecule has distinct thermophoretic properties , which are determined by its size , charge , and hydration shell .
binding of ligands typically change at least one of these parameters , resulting in changes in the thermophoretic movement of the molecule .
the change in thermophoresis can be used to derive dissociation constants ( kd ) within minutes by sequentially scanning capillaries with varying ligand concentrations .
mst has been shown to be well suited to detect binding of small molecules , fragments , or even ions to biomolecules , thus making this technique applicable to fbld . moreover , since movement of fluorescent molecules through a detection volume is monitored over time , additional information about protein aggregation and denaturation can be derived from the shape of mst traces .
here we report an automated , affinity - based fbld approach by mst to screen a library containing 193 fragments targeted against mitogen / extracellular signal - regulated kinase ( mek1 ) from the mapk signaling cascade .
mek1 is responsible for the phosphorylation and activation of downstream erk proteins , thereby regulating proliferation , differentiation , transcription regulation , and development .
aberrant signaling in this pathway is associated with unregulated cell growth , and targeting this cascade has become a viable means of developing anticancer therapies .
the library used for screening was designed by a target - restrained virtual screening campaign , so that a large number of binding fragments was expected .
the results presented here demonstrate that mst generates quantitative data for affinity rankings in a rapid and precise manner .
fourteen out of 19 fragments subjected to crystallization studies within the mst top-25 affinity ranking were shown to bind to mek1 by x - ray crystallography .
in addition , mst identified protein aggregating effects asserted by a number of fragments , thereby preventing a carryover of false positive into later stages of lead development .
thus , the use of mst in fbld represents a significant advance toward a fast , reliable , and cost - effective approach to identify and develop fragment - based leads .
the protein construct of mek1 ( residues 37383 ) was purchased from crelux ( crelux gmbh , martinsried , germany ) .
fluorescence labeling of mek1 was performed following the protocol for n - hydroxysuccinimide ( nhs ) coupling of the dye nt647 ( nanotemper technologies , munich , germany ) to lysine residues .
briefly , 100 l of a 20 m solution of mek1 protein in labeling buffer ( 130 mm nahco3 , 50 mm nacl , ph 8.2 ) was mixed with 100 l of 60 m nt647-nhs fluorophore ( nanotemper technologies ) in labeling buffer and incubated for 30 min at room temperature ( rt ) .
unbound fluorophores were removed by size - exclusion chromatography with mst buffer ( 50 mm tris , ph 7.8 , 150 mm nacl , 10 mm mgcl2 ) as running buffer .
the degree of labeling was determined using extinction coefficients 280 = 20,800 m cm for mek1 and 653 = 250,000 m cm for the nt647 fluorophore , with a correction factor ccorr of 0.031 at 280 nm , using cprotein = a280 
 
( a653*ccorr)/280 and cfluorophore = a653/653 , and was between 0.9 and 1.1 throughout all labeling reactions . for storage ,
prior to mst experiment , aliquots of nt647-mek1 were thawed on ice and centrifuged for 15 min at 4 c and 23,000  g to remove protein aggregates . to compare stability of nt647-mek1 and unmodified mek1 protein ,
thermal unfolding profiles of the proteins were recorded using the prometheus nt.48 instrument ( nanotemper technologies ) . for this , 30 l of a 2 m solution of each protein in assay buffer was prepared , and 3  10 l was loaded into nanodsf grade standard capillaries ( nanotemper technologies ) for triplicate measurements .
thermal unfolding of triplicates was analyzed in a thermal ramp from 25 to 80 c with a heating rate of 1 c / min . unfolding transition temperatures ( tm )
pretests using premium - coated and standard treated mst capillaries ( nanotemper technology ) were performed to test for adsorption of nt647 mek1 to capillary walls by analyzing capillary scans recorded by the monolith nt.115 prior to mst experiments .
mek1 did not adsorb to capillary walls in mst buffer , including 0.05% pluronic f127 ( sigma - aldrich , st .
louis , mo ) , 5 mm dtt , and 5% dmso , but strongly adsorbed to hydrophobic and standard treated capillaries in the absence of pluronic f127 .
moreover , in the absence of pluronic f127 , reproducibility of mst signals was low , and aberrant mst traces occurred , pointing toward aggregation of the protein . for subsequent experiments , standard treated capillaries and mst buffer with 0.05% pluronic f127 , 5% dmso , and 5 mm dtt ( assay buffer )
nt647-mek1 was established on a monolith nt.115 instrument ( nanotemper technologies ) and was used as a positive control throughout the screening .
for this , atp serial dilutions where prepared in assay buffer and mixed 1:1 with a solution of 3050 nm nt647-mek1 to yield a final volume of 20 l per dilution .
the reaction mixtures were loaded into standard treated capillaries and subsequently analyzed by mst at 20% and 80% mst power , respectively , and a light - emitting diode ( led ) intensity of 30% .
analysis of the interaction by thermophoresis after either 30 s laser - on time at mst 20% or 5 s laser - on time at mst 80% yielded similar kd values with similar signal - to - noise levels , so that a measurement protocol for the screening with 80% and analysis of binding after 5 s laser - on time was chosen to minimize measurement time
. stability and reproducibility of the interaction were tested by remeasuring atp binding experiments after a 2h incubation time in capillaries at rt . here ,
no change in fluorescence intensity , protein adsorption , binding amplitude , or kd value was observed , showing that the interaction was robust , and thus a suitable positive control for the screening campaign .
fragment stocks ( 100 mm ) in dmso were diluted into assay buffer to reach a final concentration of 10 mm .
subsequent liquid handling steps were carried out using a microlab starlet liquid handling system ( hamilton robotics , bonaduz , switzerland ) , modified with a multititer plate ( mtp ) turn - and - tilt station ( nanotemper technologies ) and core and iswap grippers ( hamilton robotics , bonaduz , switzerland ) for capillary chip and mtp handling . fragment predilutions were prepared for mst experiments by 12-fold 1:2 serial dilutions in assay buffer containing 10% dmso in greiner white nonbinding 384-well plates ( greiner bio - one , frickenhausen , germany ) to yield final volumes of 10 l .
nt647-mek1 stocks were centrifuged for 15 min at 23,000  g to remove aggregates , and the supernatant was subsequently transferred to the liquid handling system and diluted into assay buffer without dmso to reach a final nt647-mek1 concentration of 60 nm .
nt647-mek1 solution ( 10 l ) was then added to the fragment dilutions in the plate and mixed carefully by pipetting up and down five times to reach a final nt647-mek1 concentration of 30 nm , a final dmso concentration of 5% , and a final reaction volume of 20 l . for mst experiments ,
four rows with a total of eight 12-fold dilution series were prepared just in time before the measurement . from these four rows ,
four capillary chips with standard treated capillaries ( nanotemper technologies ) were filled with two dilution series per chip by automated dipping of the capillaries into each row of the multiwell plates .
four loaded chips were then transferred to a monolith nt.automated instrument ( nanotemper technologies ) , and the mst for each capillary was recorded at mst 80% with a laser - on time of 5 s , thereby producing eight binding curves per run , with run times of ~16 min .
over the time course of the screening , a total of 13 positive controls using the nt647-mek1-atp interaction was performed ( approximately 1 positive control every two runs ) .
additionally , a subset of fragments was investigated with laser - on times of 20 s ( shown in fig .
kd values were calculated from fragment concentration - dependent changes in normalized fluorescence ( fnorm ) of nt647-mek1 after 5 s of thermophoresis based on the law of mass action using the nt.affinity analysis software . in case fragments caused a concentration - dependent decrease in nt647-mek1 fluorescence in the serial dilution , a 
sd test  was performed to discriminate between binding - specific fluorescence quenching and loss of fluorescence due to protein precipitation . for this
, mixtures containing the  unbound  and  bound  states of nt647-mek1 ( 0 and 1 mm of the fragments , respectively ) were first centrifuged for 10 min at 23,000  g to remove protein precipitate , and then mixed with a 2 solution containing 4% sds and 40 nm dtt and heated to 95 c for 5 min to denature nt647-mek1 .
subsequently , the nt647 fluorescence of the bound and unbound samples was detected using a monolith nt.115 .
if fluorescence loss in the serial dilution is caused by binding - induced quenching , fluorescence intensities of the bound and unbound samples should be identical after denaturation ; in the case of protein loss due to precipitation , the fluorescence difference persists after the sd test , which was true for the tested fragments . for exemplary single - dose mst analysis , fnorm values at a single fragment concentration of 150 m
were calculated by subtracting the fnorm value at 150 m from the averaged fnorm values of the three lowest fragment concentrations ( 0.150.61 m , representing the unbound state ) for each serial dilution .
note that typical single - dose screenings by mst should include duplicate measurements of the fragments and dmso references on each capillary chip .
the protein construct of mek1 ( residues 37383 ) was purchased from crelux ( crelux gmbh , martinsried , germany ) .
fluorescence labeling of mek1 was performed following the protocol for n - hydroxysuccinimide ( nhs ) coupling of the dye nt647 ( nanotemper technologies , munich , germany ) to lysine residues .
briefly , 100 l of a 20 m solution of mek1 protein in labeling buffer ( 130 mm nahco3 , 50 mm nacl , ph 8.2 ) was mixed with 100 l of 60 m nt647-nhs fluorophore ( nanotemper technologies ) in labeling buffer and incubated for 30 min at room temperature ( rt ) .
unbound fluorophores were removed by size - exclusion chromatography with mst buffer ( 50 mm tris , ph 7.8 , 150 mm nacl , 10 mm mgcl2 ) as running buffer .
the degree of labeling was determined using extinction coefficients 280 = 20,800 m cm for mek1 and 653 = 250,000 m cm for the nt647 fluorophore , with a correction factor ccorr of 0.031 at 280 nm , using cprotein = a280 
 
( a653*ccorr)/280 and cfluorophore = a653/653 , and was between 0.9 and 1.1 throughout all labeling reactions . for storage ,
prior to mst experiment , aliquots of nt647-mek1 were thawed on ice and centrifuged for 15 min at 4 c and 23,000  g to remove protein aggregates .
to compare stability of nt647-mek1 and unmodified mek1 protein , thermal unfolding profiles of the proteins were recorded using the prometheus nt.48 instrument ( nanotemper technologies ) . for this , 30 l of a 2 m solution of each protein in assay buffer was prepared , and 3  10 l was loaded into nanodsf grade standard capillaries ( nanotemper technologies ) for triplicate measurements .
thermal unfolding of triplicates was analyzed in a thermal ramp from 25 to 80 c with a heating rate of 1 c / min . unfolding transition temperatures ( tm )
pretests using premium - coated and standard treated mst capillaries ( nanotemper technology ) were performed to test for adsorption of nt647 mek1 to capillary walls by analyzing capillary scans recorded by the monolith nt.115 prior to mst experiments .
mek1 did not adsorb to capillary walls in mst buffer , including 0.05% pluronic f127 ( sigma - aldrich , st .
louis , mo ) , 5 mm dtt , and 5% dmso , but strongly adsorbed to hydrophobic and standard treated capillaries in the absence of pluronic f127 .
moreover , in the absence of pluronic f127 , reproducibility of mst signals was low , and aberrant mst traces occurred , pointing toward aggregation of the protein . for subsequent experiments , standard treated capillaries and mst buffer with 0.05% pluronic f127 , 5% dmso , and 5 mm dtt ( assay buffer ) were used . the interaction between adenosine triphosphate ( atp ) and nt647-mek1
was established on a monolith nt.115 instrument ( nanotemper technologies ) and was used as a positive control throughout the screening . for this , atp serial dilutions where prepared in assay buffer and mixed 1:1 with a solution of 3050 nm nt647-mek1 to yield a final volume of 20 l per dilution .
the reaction mixtures were loaded into standard treated capillaries and subsequently analyzed by mst at 20% and 80% mst power , respectively , and a light - emitting diode ( led ) intensity of 30% .
analysis of the interaction by thermophoresis after either 30 s laser - on time at mst 20% or 5 s laser - on time at mst 80% yielded similar kd values with similar signal - to - noise levels , so that a measurement protocol for the screening with 80% and analysis of binding after 5 s laser - on time was chosen to minimize measurement time
. stability and reproducibility of the interaction were tested by remeasuring atp binding experiments after a 2h incubation time in capillaries at rt . here , no change in fluorescence intensity , protein adsorption , binding amplitude , or kd value was observed , showing that the interaction was robust , and thus a suitable positive control for the screening campaign .
fragment stocks ( 100 mm ) in dmso were diluted into assay buffer to reach a final concentration of 10 mm .
subsequent liquid handling steps were carried out using a microlab starlet liquid handling system ( hamilton robotics , bonaduz , switzerland ) , modified with a multititer plate ( mtp ) turn - and - tilt station ( nanotemper technologies ) and core and iswap grippers ( hamilton robotics , bonaduz , switzerland ) for capillary chip and mtp handling . fragment predilutions were prepared for mst experiments by 12-fold 1:2 serial dilutions in assay buffer containing 10% dmso in greiner white nonbinding 384-well plates ( greiner bio - one , frickenhausen , germany ) to yield final volumes of 10 l .
nt647-mek1 stocks were centrifuged for 15 min at 23,000  g to remove aggregates , and the supernatant was subsequently transferred to the liquid handling system and diluted into assay buffer without dmso to reach a final nt647-mek1 concentration of 60 nm .
nt647-mek1 solution ( 10 l ) was then added to the fragment dilutions in the plate and mixed carefully by pipetting up and down five times to reach a final nt647-mek1 concentration of 30 nm , a final dmso concentration of 5% , and a final reaction volume of 20 l . for mst experiments ,
four rows with a total of eight 12-fold dilution series were prepared just in time before the measurement . from these four rows ,
four capillary chips with standard treated capillaries ( nanotemper technologies ) were filled with two dilution series per chip by automated dipping of the capillaries into each row of the multiwell plates .
four loaded chips were then transferred to a monolith nt.automated instrument ( nanotemper technologies ) , and the mst for each capillary was recorded at mst 80% with a laser - on time of 5 s , thereby producing eight binding curves per run , with run times of ~16 min . over the time course of the screening ,
a total of 13 positive controls using the nt647-mek1-atp interaction was performed ( approximately 1 positive control every two runs ) .
additionally , a subset of fragments was investigated with laser - on times of 20 s ( shown in fig .
kd values were calculated from fragment concentration - dependent changes in normalized fluorescence ( fnorm ) of nt647-mek1 after 5 s of thermophoresis based on the law of mass action using the nt.affinity analysis software . in case fragments caused a concentration - dependent decrease in nt647-mek1 fluorescence in the serial dilution , a 
sd test  was performed to discriminate between binding - specific fluorescence quenching and loss of fluorescence due to protein precipitation .
for this , mixtures containing the  unbound  and  bound  states of nt647-mek1 ( 0 and 1 mm of the fragments , respectively ) were first centrifuged for 10 min at 23,000  g to remove protein precipitate , and then mixed with a 2 solution containing 4% sds and 40 nm dtt and heated to 95 c for 5 min to denature nt647-mek1 .
subsequently , the nt647 fluorescence of the bound and unbound samples was detected using a monolith nt.115 .
if fluorescence loss in the serial dilution is caused by binding - induced quenching , fluorescence intensities of the bound and unbound samples should be identical after denaturation ; in the case of protein loss due to precipitation , the fluorescence difference persists after the sd test , which was true for the tested fragments . for exemplary single - dose mst analysis , fnorm values at a single fragment concentration of 150 m
were calculated by subtracting the fnorm value at 150 m from the averaged fnorm values of the three lowest fragment concentrations ( 0.150.61 m , representing the unbound state ) for each serial dilution .
note that typical single - dose screenings by mst should include duplicate measurements of the fragments and dmso references on each capillary chip .
for mst - based fbld , we designed an automated workflow  including the preparation of serial dilutions , filling of capillary chips , and initiation of mst experiments  to quantify the binding of 193 fragments to purified mek1 protein ( fig .
lysine residues on mek1 were fluorescently labeled with the fluorophore nt647 , and the protein was subsequently tested in thermal unfolding experiments , verifying that the fluorescent label did not compromise protein stability ( suppl .
optimal buffer conditions were determined in pretests that evaluated mst signal reproducibility and the suppression of unspecific adsorption to capillary walls ( fig .
nt647-mek1 functionality was confirmed by quantifying its mg - dependent interaction with atp by mst ( fig .
1d ) , yielding kd values of 4.5 m in the presence and 140 m in the absence of mgcl2 , respectively , which is in good agreement with previous results . reproducibility and long - term stability of the nt647-mek1-atp interaction were confirmed ( suppl .
3b ) , and this interaction was subsequently used as a positive control throughout the mst screening .
( b ) automated mst setup using a hamilton starlet liquid handling station in conjunction with a monolith nt.automated instrument . using a standard mtp gripper ( a )
, capillary chips can be loaded with solutions containing fluorescent target molecule and fragment dilutions in 384-well plates ( b ) .
the filled chips are then transferred to the tray of the mst instrument ( c ) and mst experiments are initiated .
the scheme on the right illustrates the rationale of the adsorption test , in which higher fluorescence intensity on the capillary walls results in a distorted capillary shape profile .
( d ) binding of atp to nt647-labeled mek1 in the presence and absence of mgcl2 determined by mst .
( e ) tabular overview of duration and sample consumption of the affinity screening by mst .
( g ) mst binding curves of the top-25 fragment hits and the internal atp positive control ( red ) . for screening , 12-fold serial dilutions of fragments in assay buffer and subsequent mixing with nt647-mek1
after preparation of serial dilutions , capillary chips were automatically filled using a standard mtp gripper and then transferred to the capillary chip tray of the integrated mst instrument ( fig .
mst experiments were initiated automatically and eight kd values were determined per run ( two serial dilutions per chip ) .
the mst - based kd determination for 193 fragments was completed within < 7 h , consuming only 90 g of mek1 protein ( fig .
1e ) , demonstrating the fast and economical nature of this fragment screening approach . from 193 analyzed fragments ,
73 ( 37.8% ) hits were found to interact with mek1 by mst with different affinities ( fig .
1f , g ) , as indicated by fragment concentration - dependent changes in the thermophoretic movement of nt647-mek1 .
the relatively high hit rate reflects the preselection of the library by virtual screen for fragments that specifically bind to the atp binding cleft of mek1 .
the hit rate determined by mst is higher than those determined by other biophysical techniques for the same library on mek1 , highlighting the technique s ability to identify a larger pool of chemical matter that could be explored at the start of a hit - to - lead process . from the 73 fragments that induced mst responses
response curves with clearly defined bound and unbound states and kd values of < 200 m , and were classified as best hits ( fig .
in addition to standard dose  response binding experiments , specificity of the interaction of the top-25 fragments with the atp binding cleft of mek1 was confirmed by obliteration of the binding signal in competition experiments using binding buffer with 5 mm atp ( not shown ) .
we next investigated how mst - derived data correlated with results derived from orthogonal methods .
the mst affinity ranking showed a good correlation with a qualitative dsf ranking , which monitors shifts in the unfolding transition temperature ( tm ) of mek1 upon ligand binding .
however , correlation between spr and dsf results , as well as the correlation between spr and mst results , was significantly lower ( fig .
interestingly , a correlation trend was detected between kd values and tm values determined by dsf ( suppl .
4b ) , similar to previously reported observations . however , one potential limitation of dsf is that ligand - induced tm shifts can be small ( < 1 c ) , as in the present case of mek1 , and affinities have to be relatively high ( kd < 50 m ) to register robust tm shifts , which limits its applicability for low - affinity fragment screens .
consequently , only 12 fragments that increased the unfolding temperature of mek by 0.8 c were declared clear binders and transferred to x - ray analysis , disregarding a large number of potential false negatives . by comparison , mst enabled 73 fragments to be ranked based on their true binding affinity .
an additionally performed single - dose analysis of the mst data showed that a screening with a single - fragment concentration of 150 m would have been sufficient to robustly identify the x - ray confirmed hits ( on the expense of affinity information ) , while reducing measurement time by sixfold to ~70 min ( suppl .
, single - dose mst hits yielded an excellent overlap with dsf , but not with spr hits ( suppl .
( a ) tabular overview of the top-25 fragments from the mst - based kd screen in comparison to results obtained by dsf , spr , and x - ray crystallography .
( b ) venn diagram depicting distribution and overlap of hits determined by dsf , spr , and mst
numbers in overlapping regions correspond to shared hits of two or all three methods , respectively .
( c ) summary of x - ray - confirmed fragment hits determined by dsf , spr , and mst .
seven out of eight previously characterized x - ray positive binders were among the top-15 fragments ranked based on their mst affinity ( fig .
in order to test whether mst - identified binders yield complex structures with mek1 , we analyzed an additional 11 mst positive fragments from the top-25 mst hits by x - ray crystallography .
binding of 7 out of these 11 fragments to mek1 could be unambiguously confirmed , as clear electron densities with defined orientations in the atp binding site of mek1 were detectable ( fig .
3 ; other structures not shown due to proprietary reasons ) . notably , even the low - affinity binder , # 132 , could be identified based on dose - dependent mst signal changes ( mst rank 65 ; suppl .
4c ) , demonstrating that mst can detect and quantify interactions over a wide concentration range if dose 
the ability to detect low - affinity binders is particularly important for fragment screening campaigns , since the initial affinity of a fragment does not necessarily correlate with the potency of a mature compound , so that weak binders  which are potentially omitted by other biophysical techniques  can be of high value for the subsequent lead generation process .
protein hinge and dfg motif are highlighted in green and pink , respectively ( pdb : 5bx0 ) .
( b ) fragment 92 chemical formula and detailed view of the binding mode of compound 92 .
other important aspects of fragment screening campaigns comprise the exclusion of false positives and false negatives .
mst provides a direct feedback on ligand - induced aggregation and other secondary effects that are identified based on aberrant mst traces . from the 193 investigated fragments , 81 reduced tm by more than 0.4 c as determined by dsf ( fig .
mst initially identified 5 ( 20% of all top-25 mst hits ) as potential binders , while spr identified 14 ( 47% of all spr hits ) of those fragments as potential binders ( suppl .
closer inspection of mst data , however , showed that four out of the five potential hits with destabilizing properties showed clearly aberrant mst traces , pointing toward protein aggregation and/or denaturation ( fig .
2a ) . while mst data from regular binders show normal mst traces and a constant target fluorescence with a variance of < 10% throughout the serial dilution ( fig .
4b ) , several fragments , including fragments # 59 , # 106 , and # 186 , introduced irregular , bumpy mst traces at high concentrations , which are caused by protein aggregates moving through the temperature gradient ( fig .
4c ) . notably , fragment # 55 stabilized mek1 in dsf by 0.4 c , but caused irregular mst traces and also showed no binding to mek1 in x - ray analysis , suggesting that this fragment causes protein aggregation without destabilizing mek1 .
aggregating effects could also be identified for fragments outside of the top-25 mst ranking , which reduced tm by dsf ( fig .
the integration of algorithms that automatically detect and quantify the aggregation of target molecules from mst traces would allow for quickly identifying and excluding aggregation - inducing fragments and compounds . besides aberrant mst traces , several fragments , including fragments # 152 and # 193 , induced a ligand concentration - dependent drop in the fluorescence intensity of nt647-mek1 ( fig .
4d ) , indicating protein loss , for example , due to protein unfolding and adsorption to multiwell plate walls or pipette tip surfaces , which could be confirmed in subsequent tests .
( a ) plot of changes in unfolding transition temperature tm by dsf vs. mst rank .
( b ) example of regular fluorescence and mst traces of nt647 mek1 in a serial dilution upon binding to fragment # 92 .
( upper panel ) initial fluorescence values in capillaries do not differ by more than 10% throughout the serial dilution .
( lower panel ) plot of the normalized fluorescence over time of mst experiments with nt647-mek1 and fragment # 92 .
regular mst traces have a smooth appearance and show a ligand concentration - dependent shift in magnitude .
( c ) examples for fragment - induced aberrant mst traces due to protein aggregation / denaturation at elevated fragment concentration . for each fragment ,
three or four capillaries at the highest fragment concentrations ( 50.625 mm ) are shown .
( d ) examples for fragment concentration - dependent loss of fluorescence intensity throughout the serial dilution . fragments # 152 and # 193 reduce the initial fluorescence intensity by 86% and 42% , respectively , which could be attributed to protein loss .
the exclusion of mst - identified promiscuous binders from further analysis improved the hit - to - structure ratio from the crystallized fragments from 73% to 86% ( suppl .
thus , additional mst - derived information about protein quality can be used as a filter to increase the hit rate of the screening , and to prevent destabilizing / aggregating fragments from entering the hit - to - lead stage of drug development . in conclusion ,
mst robustly identified , quantified , and validated a set of fragment hits with micromolar affinity for mek1 , which could be followed up in hit - to - lead development .
moreover , mst provided additional information about unwanted secondary effects of fragments on protein integrity that were not detected by other methods , and which would have prevented false - positive hits from entering later stages of hit expansion , as well as rescuing fragments classified as false negatives .
furthermore , the primary hits identified by mst would have provided increased chemical depth of compounds forwarded for hit - to - lead expansion . combining speed and low material requirements with the reliable identification of true binders by deriving binding affinities ,
mst has been demonstrated to be an effective method for screening fragments . used in conjunction with other biophysical techniques , mst has the potential to significantly improve fbld workflows .
 fragment - based lead discovery has proved to be an effective alternative to high - throughput screenings in identifying chemical matter that can be developed into robust lead compounds . 
 the search for optimal combinations of biophysical techniques that can correctly and efficiently identify and quantify binding can be challenging due to the physicochemical properties of fragments . in order to minimize the time and costs of screening , 
 optimal combinations of biophysical techniques with maximal information content , sensitivity , and robustness are needed . here 
 we describe an approach utilizing automated microscale thermophoresis ( mst ) affinity screening to identify fragments active against mek1 kinase . 
 mst identified multiple hits that were confirmed by x - ray crystallography but not detected by orthogonal methods . 
 furthermore , mst also provided information about ligand - induced aggregation and protein denaturation . 
 the technique delivered a large number of binders while reducing experimentation time and sample consumption , demonstrating the potential of mst to execute and maximize the efficacy of fragment screening campaigns . 
Introduction
Material and Methods
Protein Labeling
Protein Thermal Stability Measurements
Assay Development for MST Screening
MST Fragment Screening
Results and Discussion
Supplementary Material