molecules

Article Application of a Substrate-Mediated Selection with c-Src Tyrosine Kinase to a DNA-Encoded Chemical Library

1,2, 1, 1 1,3 1 1 Dongwook Kim †, Yixing Sun †, Dan Xie , Kyle E. Denton , Hao Chen , Hang Lin , Michael K. Wendt 1, Carol Beth Post 1 and Casey J. Krusemark 1,* 1 Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University Center for Cancer Research, Purdue University, West Lafayette, IN 47907, USA 2 Dencoda, LLC, West Lafayette, IN 47906, USA 3 X-Chem Pharmaceuticals, Waltham, MA 02453, USA * Correspondence: [email protected] These authors contributed equally to this work. †


Received: 24 June 2019; Accepted: 26 July 2019; Published: 30 July 2019


Abstract: As aberrant activity of protein kinases is observed in many disease states, these enzymes are common targets for therapeutics and detection of activity levels. The development of non-natural protein kinase substrates offers an approach to protein substrate competitive inhibitors, a class of kinase inhibitors with promise for improved specificity. Also, kinase activity detection approaches would benefit from substrates with improved activity and specificity. Here, we apply a substrate-mediated selection to a peptidomimetic DNA-encoded chemical library for enrichment of molecules that can be phosphorylated by the protein tyrosine kinase, c-Src. Several substrates were identified and characterized for activity. A lead compound (SrcDEL10) showed both the ability to serve as a substrate and to promote ATP hydrolysis by the kinase. In inhibition assays, compounds displayed µ IC500 s ranging from of 8–100 M. NMR analysis of SrcDEL10 bound to the c-Src:ATP complex was conducted to characterize the binding mode. An ester derivative of the lead compound demonstrated cellular activity with inhibition of Src-dependent signaling in cell culture. Together, the results show the potential for substrate-mediated selections of DNA-encoded libraries to discover molecules with functions other than simple protein binding and offer a new discovery method for development of synthetic tyrosine kinase substrates.

Keywords: DNA-encoded chemical library; protein tyrosine kinases; substrate-mediated selection; c-Src

1. Introduction DNA-encoded chemical libraries (DELs) have recently become a widespread approach for the discovery of new protein ligands [1,2]. DELs offer advantages in cost, throughput, and access to greater chemical space over conventional de novo discovery by high throughput screening. Discovery of several new inhibitors to many classes of therapeutically relevant protein targets has now been reported [3]. These include molecules currently in clinical trials. The predominant approach to ligand discovery from DELs is an affinity-mediated selection assay, whereby DNA-linked ligands are enriched from the library through binding to a protein target immobilized on a solid support [4]. While successful in many cases, these assays can suffer from drawbacks, such as background binding to the support matrix, limited control of protein concentration, and the potential for loss of protein activity with immobilization. Also, by design, affinity-mediated selections enrich simply for the function of binding. While seldom reported or investigated thoroughly,

Molecules 2019, 24, 2764; doi:10.3390/molecules24152764 www.mdpi.com/journal/molecules Molecules 2019, 24, 2764 2 of 19 such selections can result in so called “innocent binders”, which do bind the protein but do not inhibit the desired protein activity. Thus, an oft-cited goal for DEL assays is to develop selections to yield molecules with functions beyond simple protein binding, with the hope that all hit molecules will inhibit a desired activity of the protein target. One approach to this is to perform parallel selections in the presence or absence of known protein binders (inhibitors or substrates) to illuminate the binding mode of hit ligands [5]. Another approach is the release of small molecules from bead-bound DELs within confined droplets for colorimetric inhibition assays in microfluidic devices [6]. Yet another approach is a substrate-based selection, which can enrich molecules on the basis of enzymatic turnover [7]. Substrate selections have seen application in both phage display and mRNA display [8,9], but have seen limited application with DELs [10]. Here, we employ a substrate-mediated selection for selecting phospho-accepting substrates of the tyrosine kinase c-Src, that could then serve as protein substrate competitive inhibitors. Not only should such a selection yield hits that bind specifically at the protein substrate site, but it should also be less prone to non-specific interactions than could enrich molecules in a typical affinity-mediated selection. A substrate-mediated selection also has greater potential for sensitivity in enriching low affinity hits as it can take advantage of enzymatic turnover (much like an ELISA), and therefore it has a lower requirement for specific activity of the protein target, which can be problematic for protein targets with poor stability. Protein kinases are well-validated drug targets and the second largest drug target class [11]. Yet, there remains a critical need for kinase inhibitors that are potent, highly specific, and not susceptible to drug resistance mutations. Nearly all reported kinase inhibitors and the vast majority of the 37 clinically-approved therapeutics target the ATP binding site [12]. With ~518 protein kinases utilizing ATP as a substrate, achieving specific inhibition with ATP competitive inhibitors is a challenge [13]. Strategies that exploit less-conserved residues adjacent to the ATP-binding site have improved selectivity, yet arising mutations to these residues can abolish drug binding with minimal effects on kinase enzymatic function. For example, a “gatekeeper” mutation (T315I) in Bcr-Abl leads to imatinib resistance but shows little effect on the binding of ATP [11]. The development of such resistance mutations is a major problem with kinase inhibitors used in cancer treatment. Rather than targeting inhibitors to areas distal from the active site to achieve selectivity, targeting sites most critical to kinase activity will more likely yield inhibitors recalcitrant to resistance, as such mutants are likely to be non-functional. In addition, ATP-competitive inhibitors have a very high requirement for affinity to achieve cellular activity due to the typical Km values for ATP being low to mid- micromolar and cellular ATP levels being low millimolar [14]. The potency required for efficacy with ligands targeting the protein substrate site is much lower, since substrates in cells are generally present at concentrations near or below their Km [15]. While kinase substrate interactions are often mediated by additional proteins in vivo, kinases possess a great deal of “naked” substrate specificity. Optimal peptide substrates, determined with purified kinases in in vitro assays, vary in sequence considerably among kinases [16]. Kinase residues in the substrate binding site are much less conserved than the ATP site [17]. While in vitro studies with kinases have shown overlap in peptide substrate sequence, there are clear, distinct substrate preferences, even among highly related kinases, which demonstrates potential for differentiation [18]. Generally, protein tyrosine kinases prefer peptide substrates with multiple negatively charged amino acids [19]. Optimization of natural peptide substrates for several non-receptor tyrosine kinases found an optimal 1 sequence for c-Src of EEEIYGIFG (kcat = 2.9 s− ,Km = 14 µM). Prior work from Lam and coworkers generated efficient substrates for c-Src that lack acidic residues completely, which demonstrates high plasticity in substrate recognition. These include sequences GIYWHHY (Km = 21 µM) and YIYGSFK (Km = 55 µM) [20]. These peptides have been converted into pseudosubstrate inhibitors with low µM

IC500 s [21]. Molecules 2019, 24, 2764 3 of 19

Prior work has demonstrated that even homologous kinases can be differentially inhibited through pharmacological targeting at the protein-substrate site [22]. For example, work with modified peptide substrates of the PKC family generated inhibitors with remarkable potency and selectivity [23]. Work from Soellner and coworkers presented a substrate-based fragment screening approach for discovery of protein substrate competitive ligands [24]. This technique was successful in generating the first substrate competitive inhibitor for c-Src, which demonstrated modest potency (16 µMKi), yet excellent selectivity among Src family kinases and activity in cell culture. Additionally, work with Src and DNA-encoded peptide macrocycles yielded inhibitors that exhibited bisubstrate-competitive behavior. Interestingly, these molecules inhibited substrate binding allosterically and showed excellent specificity over other Src family kinases [25]. A wide variety of techniques rely on peptides as protein kinase substrates for detection and profiling of kinase activity in biological samples. Not only are such approaches critical for drug development research [26], but they are also showing the potential utility of kinase activity as a disease biomarker in the ex vivo analysis of patient samples [27]. Examples of such methods include peptide microarrays [28], cell penetrating fluorescent sensors [29,30], and peptide reporters for detection by capillary electrophoresis [31]. Compared to the mass spectrometry-based, kinase enrichment approaches, like multiplexed inhibitor beads [32] and acyl-ATP probes [33], these methods have the advantage that activity is directly measured and not simply kinase abundance. The drawback, however, is that peptide substrates have poor specificity. In the majority of these detection approaches, non-natural substrates could substitute for natural peptide sequences directly. In addition to the potential for greater kinase selectivity, synthetic substrates could have advantages in protease stability and increased activity for improved assay sensitivity. In this work, we use the c-Src protein tyrosine kinase and a substrate-based selection for discovery from a DNA-encoded library (DEL) of peptidomimetics. We present a DEL designed to target the protein substrate site that contains natural peptides, non-natural peptides, and peptoid-inspired structures. Treatment of the library with ATP and Src kinase followed by an affinity selection using a non-specific phosphotyrosine-binding antibody allowed enrichment of substrate molecules.

2. Results

2.1. DNA-Encoded Chemical Library A DNA-encoded library was constructed in 4 encoding cycles using the routing-based approach of DNA-programmed combinatorial chemistry [34,35]. The majority of the library diversity was incorporated within the first three cycles yielding 3-mer oligoamides composed of 47 unique monomers, which were acylated in the fourth step with any of 4 carboxylates (Scheme1). Monomers in the first three steps included amino acids (both natural and non-natural), peptoids (N-substituted oligoglycines), and peptoid-inspired N-alkyl benzyl monomers [36,37] (Scheme1). See Tables S1 and S2 for full monomer set. These building blocks gave a full library complexity of 5.5 105 unique members. × Monomers were selected for the library to maximize both chemical diversity and reaction efficiency. Both fmoc-amino acid peptide chemistry and peptoid synthesis have been demonstrated on DNA and have shown to be quite robust [38,39]. Fmoc-amino acids included showed at least 90% acylation of a DNA-linked amine in rehearsal reactions (Tables S1 and S2) or in previous reports of on-DNA chemistry [38]. Similarly, all amines included were tested in the standard peptoid chemistry and demonstrated greater than 80% conversion in the SN2 reaction of a chloroacetamide on-DNA and at least 50% conversion in the subsequent acylation with chloroacetate. The library was prepared such that each building block was encoded by two different codons with the exception of building blocks for the fourth step, which were singly encoded. The codon sets were kept separate until after translation and then combined to give a two-fold genetic redundancy for each library member. Molecules 2019, 24, 2764 4 of 19 Molecules 2019, 24, x FOR PEER REVIEW 4 of 19

SchemeScheme 1. 1.Construction Construction ofof DNADNA encoded library with with 5.5 5.5 × 10105 unique5 unique members. members. × ThisThis library library design design waswas selectedselected to bridge the gap gap in in structure structure from from natural natural peptide peptide substrates substrates towardstowards molecules molecules withwith moremore drug-likedrug-like properties.properties. The The tertiary tertiary amides amides of of both both the the peptoid peptoid and and peptoid-inspiredpeptoid-inspired monomersmonomers areare increasinglyincreasingly hydr hydrophobicophobic compared compared to to the the secondary secondary amides amides of of peptides.peptides. Peptoids Peptoids have have shown shown markedly markedly greater greater permeability permeability than than peptides peptides in several in several studies studies [40,41 ]. Additionally,[40,41]. Additionally, several phenol several containing phenol containing building blocksbuilding were blocks included were toincluded increase to the increase likelihood the of suitablelikelihood phospho-acceptors. of suitable phospho-acceptors. Targeting the protein Targeting substrate the protein site with substrate conventional site with medicinal conventional chemistry approachesmedicinal chemistry has been challenging,approaches has and been thus challenging, it has been sparselyand thusexplored it has been [15 sparsely]. The shallow, explored exposed, [15]. andThe extended shallow, exposed, binding siteand extended of the kinase binding substrate site of the protein-protein kinase substrate interaction protein-protein is not amenableinteraction to structure-basedis not amenable design to structure-based nor is it well suited design to bindingnor is it small,well suited flat heterocycles to binding typical small, offlat high heterocycles throughput screeningtypical of libraries high throughput [42]. screening libraries [42]. As a quality control measure, the library was sequenced before and after translation. The codon As a quality control measure, the library was sequenced before and after translation. The codon abundances were shown to be narrowly distributed and did not change appreciably after translation, abundances were shown to be narrowly distributed and did not change appreciably after translation, indicating that little codon bias occurs in the process of hybridization routing and chemistry (Figure indicating that little codon bias occurs in the process of hybridization routing and chemistry (Figure S1). Based on codon abundances in the translated library, gene abundances were approximated for S1). Based on codon abundances in the translated library, gene abundances were approximated for the the 2 × 105 genes encoding the initial 3 steps, as previously described [10]. The maximum disparity in 2 105 genes encoding the initial 3 steps, as previously described [10]. The maximum disparity in gene× abundance was 100-fold and the vast majority of the library (95%) was within a 10-fold window gene(Figure abundance S1c). To wasfurther 100-fold validate and both the genetic vast majority and chemical of the library steps in (95%) library was assembly, within a we 10-fold substituted window (Figureone of S1c).the building To further blocks validate at chemical both genetic step 3 with and a chemical known pharmacophore steps in library (an assembly, aryl sulfonamide) we substituted to onecarbonic of the buildinganhydrase blocks II (CAII) at chemical and demonstrated step 3 with en arichment known pharmacophoreof ligands by DNA (an sequencing aryl sulfonamide) (Figure to carbonicS2). All anhydrase of the top II 400 (CAII) enriching and demonstrated molecules cont enrichmentained the of pharmacophore. ligands by DNA SAR sequencing of the (Figureenriched S2). Allmolecules of the top was 400 consistent enriching with molecules previous contained published the ligands pharmacophore. [43]. SAR of the enriched molecules was consistent with previous published ligands [43]. 2.2. Tyrosine Kinase Substrate-Mediated Selection 2.2. Tyrosine Kinase Substrate-Mediated Selection We employed a two-step selection strategy to enrich for molecules that can serve as substrates to Wec-Src employed (Figure a1). two-step DNA-encoded selection molecules strategy to are enrich treated for moleculeswith active that c-Src can servekinase as substratesand ATP. to c-SrcPhosphorylated (Figure1). DNA-encoded molecules are molecules then enriched are treated with a withbroad active specificity c-Src kinaseanti-phosphotyrosine and ATP. Phosphorylated antibody molecules(4G10 clone) are immobilized then enriched on Protein with a G broad magnetic specificity beads [44]. anti-phosphotyrosine This approach has been antibody applied (4G10 in phage clone) immobilizeddisplay libraries on Protein for discovery G magnetic of natural beads [peptid44]. Thise substrates approach [45]. has A been pote appliedntial drawback in phage of display the librariesapproach for is discovery that certain of non-natural natural peptide substrates substrates may not [45 be]. recognized A potential by drawback the antibody. of the Also, approach as c-Src is thathas certain been shown non-natural to phosphorylate substrates aliphatic may not oxygens be recognized [46], these by substrates the antibody. are unlikely Also, as to c-Src be enriched has been shownby this to approach. phosphorylate Given aliphaticthat phosphoprotein oxygens [46 ],an thesed phosphopeptides substrates are unlikelycan be eluted to be from enriched antibody by this approach.supports with Given phenyl that phosphoproteinphosphate [47], we and hypothesized phosphopeptides that this can antibody be eluted contains from reasonable antibody affinity supports withfor phenylsimple phosphorylated phosphate [47], wephenols. hypothesized In order thatto minimize this antibody bias in contains the selection reasonable based a onffinity variation for simple in phosphorylatedaffinity for the antibody, phenols. antibody In order tobeads minimize were used bias at in as the high selection a concentration based on as variation possible. in Selections affinity for theemploying antibody, ATP- antibodyγS as a beads co-substrate were used with at subsequent as high a concentration labeling of phosphothiolates as possible. Selections were additionally employing ATP-exploredγS as but a co-substrate were met with with several subsequent problems labeling [7,10]. ofATP- phosphothiolatesγS is a non-natural were and additionally poor substrate explored for butc-Src were [48]. met Commercially with several available problems ATP- [7,10γS]. is ATP- significantlyγS is a non-natural contaminated and with poor high substrate levels of for inhibitory c-Src [48 ]. CommerciallyADP [49]. Additionally, available ATP-we observedγS is significantly low, but signif contaminatedicant, reactivity with of high DNA levels with of both inhibitory iodoacetamide ADP [49 ]. biotin and pyridyldisulfide biotin in attempts to selectively label DNA-linked phosphothiolates and Additionally, we observed low, but significant, reactivity of DNA with both iodoacetamide biotin were concerned that certain library building blocks may have higher levels of reactivity. Thus, we and pyridyldisulfide biotin in attempts to selectively label DNA-linked phosphothiolates and were

Molecules 2019, 24, 2764 5 of 19 Molecules 2019, 24, x FOR PEER REVIEW 5 of 19 concernedfound using that the certain native library ATP substrate building with blocks the mayantibo havedy pulldown higher levels to be of preferred reactivity. and Thus, a more we robust found usingapproach. the native ATP substrate with the antibody pulldown to be preferred and a more robust approach.

FigureFigure 1. 1Src. Src kinase kinase substratesubstrate selectionselection scheme.

ToTo validate validate this this selection selection strategy, strategy, we we performed performed testtest selectionsselections using a a DNA-linked DNA-linked peptide peptide substrate.substrate. We We used used an optimalan optimal peptide peptide substrate substrate (LYNtide) (LYNtide) for Lynfor tyrosineLyn tyrosine kinase, kinase, a Src familya Src family kinase, 1 whichkinase, showed which similar showed kinetic similar parameters kinetic parameters for Src (k forcat Src= 2.0 (kcat s− =,K 2.0m s−=1, 18Kmµ =M) 18 [µM)18]. [18]. ToTo mimic mimic library library selection selection conditions, conditions, a DNA-LYNt a DNA-LYNtideide construct construct was mixed was with mixed a control with construct a control constructat a ratio at of a 1 ratio to 500. of 1This to 500. mixture This was mixture treated was at treated50 nM total at 50 DNA nM totalconcentration DNA concentration with Src kinase with and Src kinasethe selection and theselection was applied. was applied.We observed We observed robust enri robustchment enrichment (2,600-fold) (2600-fold) and recovery and recovery (9%) of (9%) the of theLYNtide LYNtide construct construct by by qPCR qPCR (Figure (Figure S3). S3). TheThe selection selection was was then then similarly similarly appliedapplied toto 99 fmolfmol ofof thethe librarylibrary (~10,000 molecules molecules of of each each member)member) together together with with a a LYNtide LYNtide construct construct doped doped inin atat approximatelyapproximately 300,000 copies. copies. In In the the library library selection,selection, the the amount amount of of enzyme enzyme used used and and incubation incubation timetime was approximately ten ten times times that that required required forfor full full phosphorylation phosphorylation of a LYNtide-DNAof a LYNtide-DNA conjugate. conjugate. Selections Selections were PCR were amplified PCR amplified using sequencing using adaptorsequencing and sample adaptor index and sample containing index primers. containing PCR primers. amplicons PCR amplicons were then were sequenced then sequenced and decoded. and DNAdecoded. sequencing DNA analysissequencing indicated analysis 2100-fold indicated enrichment 2100-foldof enrichment the LYNtide of dopethe LYNtide in, which dope was in, consistent which was consistent with assay optimization experiments by qPCR. Within the library, several high with assay optimization experiments by qPCR. Within the library, several high enriching sequences enriching sequences were observed. A cubic plot of the top 200 enriching sequences based on their were observed. A cubic plot of the top 200 enriching sequences based on their first, second, and forth first, second, and forth synthon numbers (Figures 2a, S4a) shows that the majority of these molecules synthon numbers (Figure2a, Figure S4a) shows that the majority of these molecules contain the phenol contain the phenol cap in the fourth step, which is a positive indication that tyrosine kinase substrates cap in the fourth step, which is a positive indication that tyrosine kinase substrates were enriched. were enriched. Plotting the first three synthon numbers of the molecules all containing the fourth Plotting the first three synthon numbers of the molecules all containing the fourth step phenol cap step phenol cap (Figures 2b, S4b) shows that the high enriching hits are quite distributed throughout (Figurethe library,2b, Figure while S4b) several shows of the that lower the high enriching enriching hits (~10–100-fold) hits are quite distributedoccur within throughout lines, indicating the library, these whilehits severalare among of the structurally lower enriching similar families. hits (~10–100-fold) In addition, occurnone of within the high lines, enriching indicating sequences these hits were are amongfound structurally in both of redundantly similar families. encoded In addition, libraries, nonewhich of suggests the high randomness enriching sequences in the results were likely found due in bothto ofunder redundantly sampling. encoded Recent analysis libraries, of whichDEL selection suggests sequences randomness has shown in the resultshow under likely sampling due to under can sampling.lead to low Recent reproducibility analysis of and DEL randomness selection sequences in results has [50]. shown Together, how these under results sampling suggested can lead the noise to low reproducibilitylevel in the selection and randomness was relatively in results high. [50]. Together, these results suggested the noise level in the selection24 wasmolecules relatively were high. selected for follow up on the basis of both the presence of structural similar hits24 and molecules overall enrichment were selected (at least for follow 10-fold) up (Figur on thee S5). basis To of eliminate both the false presence positives, of structural molecules similar were hitsinitially and overall synthesized enrichment on a 20-mer (at least DNA 10-fold) oligomer (Figure primer S5). Tousing eliminate identical false procedures positives, to molecules those used were in initiallylibrary synthesized construction. on These a 20-mer crude DNA products oligomer were primer placed usingon unique identical encoding procedures DNAs toby thosePCR. usedThese in libraryconstructs construction. were then These subjected crude to products the substrate- werebased placed selection on unique for hit encoding validation DNAs using by enrichment PCR. These constructsdetermined were by then qPCR subjected (Figure toS6). the Of substrate-based the molecules selectionshowing forenrichment, hit validation six were using selected enrichment for determinedsynthesis byoff qPCRencoding (Figure DNA S6). scaffolds. Of the molecules showing enrichment, six were selected for synthesis off encoding DNA scaffolds.

MoleculesMolecules2019 2019, 24, 24, 2764, x FOR PEER REVIEW 66 of of 19 19

(a)

(b)

FigureFigure 2. 2.Cubic Cubic plot plot analysis analysis of of DNA-encoded DNA-encoded chemical chemical libraries libraries (DEL) (DEL) selection. selection (.a ()a The) The highest highest 200 200 enrichingenriching members members of of the the library library encoded encoded by theby the initial initial 48 codons 48 codons (A library) (A library) are plotted are plotted indicating indicating the synthonthe synthon numbers numbers of the firstof the (x -axis),first (x second-axis), second (y-axis), ( andy-axis), fourth and (z fourth-axis) chemical (z-axis) chemical steps. (b) steps. The highest (b) The 300highest enriching 300 membersenriching ofmembers the library of encodedthe library by theencode initiald by 48 codonsthe initial (A library)48 codons that (A contained library) thethat phenolcontained cap inthe the phenol fourth cap step in arethe plottedfourth step indicating are plotted the synthonindicating numbers the synthon of the numbers first (x-axis), of the second first (x-

(yaxis),-axis), second and third (y-axis), (z-axis) and chemical third (z steps.-axis) Colorchemical of the steps. points Color corresponds of the points to the corresponds log10 of the to enrichment the log10 of relativethe enrichment to the unselected relative libraryto the unselected as indicated library in the as color indicated bar scale. in the color bar scale.

2.3.2.3. c-Src c-Src Activity Activity and and Binding Binding Assays Assays HitHit molecules molecules werewere initially characterized characterized as as substrates substrates for for c-Src c-Src using using the luciferase-based the luciferase-based ADP- ADP-GloGlo assay assay (Promega). (Promega). All six All molecules six molecules appeared appeared to be efficient to be effi substratescient substrates for Src kinase. for Src We kinase. observed We observed Km values ranging from 3–6-fold higher than the LYNtide peptide substrate, with the lowest Km values ranging from 3-6-fold higher than the LYNtide peptide substrate, with the lowest observed observed Km being 13 µM for compound SrcDEL14 (Table1). Km being 13 µM for compound SrcDEL14 (Table 1). Subsequently, we tested one of these hits, SrcDEL10, attached to a DNA scaffolds for binding affinity to Src kinase in the presence of ATP. These studies used the HPLC-purified ligand linked to 20-mer oligos, which were prepared for the prior qPCR validations under crude conditions. This construct was hybridized to a fluorescein-containing complementary oligo, and binding constants were determined by changes observed in fluorescence polarization, as previously described [51]. A binding constant of 7.5 µM was found for SrcDEL10 in the presence of ATP (Figure3a). SrcDEL10 was selected for further study based on this result and the presence of the aspartic acid monomer, which is consistent with the typically acidic, natural peptide consensus sequences.

Molecules 2019, 24, x FOR PEER REVIEW 7 of 19

Table 1. Observed values for compounds in ADP-Glo assay.

OH OH m cat cat m O HN NH K k k /K O O 2 H N N NH -1 -1 -1 H2N N O O Compound (µM) (min ) (min µM ) O NH H2N O N N OH N N H SrcDEL5 33 350 11 O O SrcDEL5 OH SrcDEL8 SrcDEL8 19 150 7.8 OH

HO SrcDEL10 18 82 4.6 O O H N 2 N O Molecules 2019, 24, 2764 O OH H 7 of 19 O N SrcDEL14 13 45 3.3 N N H N NH H 2 O Molecules 2019, 24, x FOR PEER REVIEW O O OH 7 of 19 OH O SrcDEL14 SrcDEL16 18 32 1.8 N SrcDEL10 OH Table 1. Observed values for compounds in ADP-Glo assay. OH SrcDEL21 Table28 1. Observed86 values for compounds 3.0 in ADP-Glo assay. O O O H O O OH H LYNtide N N OH N Km kcat kcat/Km H2N N H2N N m5.2 cat 390 cat m 74 O HN NH2 K k k /K O O O O H OH (DEDIYGVLP) N N N NH 1 1 1 H N N O O Compound (µM) (min-1 ) (min-1 µM-1 ) 2 HO Compound (µM) (min )− (min− µM −) O SrcDEL21 SrcDEL16NH H2N O N N OH N OH N 33 350 11 H SrcDEL5SrcDEL5 33 350 11 O O SrcDEL5 OH SrcDEL8 SrcDEL8SrcDEL8 19 19150 150 7.8 7.8 Subsequently, we tested one of these hits, SrcDEL10OH, attached to a DNA scaffolds for binding HO SrcDEL10 18 82 4.6 O 18 82 4.6 O SrcDEL10 H2N affinity to Src kinase in the presence of ATP. These studies used the HPLC-purifiedN ligandO linked to O OH H O N SrcDEL14 13 45 3.3 N N H N NH H 13 45 3.3 2 O 20-mer oligos, which were prepared for the prior qPCR OvalidationsO OH under crude conditions.OH This SrcDEL14 O SrcDEL14 SrcDEL16 18 32 1.8 N construct was hybridized to a fluorescein-containing complementarySrcDEL10 oligo,OH and binding constants SrcDEL16 18 32 1.8 SrcDEL21 28 86 3.0 OH were determined by changes observed in fluorescence polarization,O as previously described [51]. A O O O O 28 86 3.0 H H LYNtideSrcDEL21 N N N H N N binding constant of 7.5 µM was found for SrcDEL10 in2 the presence of ATPH2N (FigureN 3a). SrcDEL10 5.2 390 74 O O OH (DEDIYGVLP) N LYNtide HO was selected for further5.2 study 390 based on this 74 result and the presence of the asparticSrcDEL21 acid monomer, (DEDIYGVLP) SrcDEL16 which is consistent with the typically acidic, natural peptide consensusOH sequences.

Subsequently, we tested one of these hits, SrcDEL10, attached to a DNA scaffolds for binding affinity to Src kinase in the presence of ATP. These studies used the HPLC-purified ligand linked to 20-mer oligos, which were prepared for the prior qPCR validations under crude conditions. This construct was hybridized to a fluorescein-containing complementary oligo, and binding constants were determined by changes observed in fluorescence polarization, as previously described [51]. A binding constant of 7.5 µM was found for SrcDEL10 in the presence of ATP (Figure 3a). SrcDEL10 was selected for further study based on this result and the presence of the aspartic acid monomer, which is consistent with the typically acidic, natural peptide consensus sequences.

(a) (b)

FigureFigure 3. 3. ValidationValidation of SrcDEL10 asas a a hit hit molecule molecule from from the the substrate-mediated substrate-mediated selection. selection. (a) on-DNA(a) on-DNA bindingbinding affinity affinity to to c-Src c-Src measured measured by fluorescence fluorescence polarization inin thethe presencepresence ofof ATPATP(100 (100µ µM).M). A SrcDEL10A SrcDEL10 modifiedmodified oligo oligo was was hybridized hybridized to aa fluoresceinfluorescein amide amide (FAM) (FAM) modified modified complementary complementary oligooligo for for the the binding binding assay. ((bb)O) Off-DNAff-DNA HPLC HPLC analysis analysis of ofSrcDEL10 SrcDEL10(200 (200µM) µM) with with treatment treatment of ATP of ATP (1(1 mM) mM) and and c-Src c-Src at highhigh levelslevels (1(1µ µM,M, yellow, yellow, and and 10 10µ M,µM, red), red), which which gave gave small small amounts amounts of the of the phosphorylatedphosphorylated molecule. PeakPeak identityidentity was was verified verified by by LC LC/MS/MS analysis analysis (Figure (Figure S9). S9). First, we sought to confirm the activity of SrcDEL10 as a kinase substrate by LC/MS. Treatment of First, we sought(a )to confirm the activity of SrcDEL10 as a kinase substrate(b) by LC/MS. Treatment SrcDEL10 with high levels of enzyme (1 µM) yielded very little (~1%) of the phosphorylated compound of SrcDEL10 with high levels of enzyme (1 µM) yielded very little (~1%) of the phosphorylated (FigureFigure3 3.b). Validation Under identical of SrcDEL10 conditions, as a hit molecule the LYNtide from the peptide substrate-mediated was fully phosphorylated selection. (a) on-DNA (Figure S7). binding affinity to c-Src measured by fluorescence polarization in the presence of ATP (100 µM). A compoundThis suggested (Figure that 3b). the Under signal identical observed conditions in the ADP-Glo, the LYNtide assay was peptide likely duewas tofully ATP phosphorylated hydrolysis SrcDEL10 modified oligo was hybridized to a fluorescein amide (FAM) modified complementary (Figurerather thanS7). phosphotransfer.This suggested that To confirm the signal this, compoundsobserved in were the ADP-Glo tested with assa an NADH-based,y was likely due coupled to ATP oligo for the binding assay. (b) Off-DNA HPLC analysis of SrcDEL10 (200 µM) with treatment of ATP hydrolysisenzyme assay rather that than also detectsphosphotransfer. ADP generation To conf [52].irm For this,SrcDEL5 compounds, 8, 10, and were21, tested ADP generation with an NADH- was (1 mM) and c-Src at high levels (1 µM, yellow, and 10 µM, red), which gave small amounts of the based,observed coupled in large enzyme molar assay excess that over also the detects compound, ADP whereas generation ADP [52]. generation For SrcDEL5 for LYNtide, 8, 10, and occurred 21, ADP phosphorylated molecule. Peak identity was verified by LC/MS analysis (Figure S9). generationat approximately was observed one equivalent in large (Figure molar S8). excess This over additionally the compound, supported whereas that the ADP-GloADP generation activity for LYNtide occurred at approximately one equivalent (Figure S8). This additionally supported that the observedFirst, we was sought due to to confirm activation the activity of ATP hydrolysisof SrcDEL10 and as a not kinase phosphotransfer substrate by LC/MS. to these Treatment compounds. ADP-Glo activity observed was due to activation of ATP hydrolysis and not phosphotransfer to these of SrcDEL10For SrcDEL14 withand high16 ,levels little turnover of enzyme was (1 observed. µM) yielded very little (~1%) of the phosphorylated compoundcompounds.We (Figure prepared For 3b).SrcDEL14 10 Under derivatives andidentical 16 of, littleSrcDEL10 conditions turnovero, ffthe-DNA was LYNtide observed. to explore peptide structure was fully activity phosphorylated relationships (Figure(Figure S7).4). This Initially, suggested these were that each the assessedsignal obse as substratesrved in the in ADP-Glo the ADP-Glo assa assayy was (Table likely S3). due Surprisingly, to ATP hydrolysisthe assay rather indicated than phosphotransfer. that 10–6, which lacksTo conf airm phenolic this, compounds oxygen, was were a comparable tested with substrate an NADH- to the based,parental coupled compound enzyme (Figureassay that5a). also Compound detects ADP10– generation8, which lacks [52]. onlyFor SrcDEL5 the cyclopropyl, 8, 10, and group, 21, ADP lacked generationactivity altogether.was observed These in resultslarge molar were potentiallyexcess over inconsistent the compound, with bothwhereas the enrichment ADP generation of SrcDEL10 for LYNtide occurred at approximately one equivalent (Figure S8). This additionally supported that the ADP-Glo activity observed was due to activation of ATP hydrolysis and not phosphotransfer to these compounds. For SrcDEL14 and 16, little turnover was observed.

Molecules 2019, 24, x FOR PEER REVIEW 8 of 19

We prepared 10 derivatives of SrcDEL10 off-DNA to explore structure activity relationships (Figure 4). Initially, these were each assessed as substrates in the ADP-Glo assay (Table S3). Surprisingly, the assay indicated that 10–6, which lacks a phenolic oxygen, was a comparable substrate to the parental compound (Figure 5a). Compound 10–8, which lacks only the cyclopropyl group, lacked activity altogether. These results were potentially inconsistent with both the enrichment of SrcDEL10 from the library by binding of the phosphorylated molecule to the 4G10 Molecules 2019, 24, x FOR PEER REVIEW 8 of 19 antibody and with the activity observed in the ADP-Glo assay being from a phosphotransfer to the small molecule. We prepared 10 derivatives of SrcDEL10 off-DNA to explore structure activity relationships (Figure 4). Initially, these were each assessed as substrates in the ADP-Glo assay (Table S3). Surprisingly, the assay indicated that 10–6, which lacks a phenolic oxygen, was a comparable substrate to the parental compound (Figure 5a). Compound 10–8, which lacks only the cyclopropyl group,Molecules lacked2019, 24 activity, 2764 altogether. These results were potentially inconsistent with both8 of 19the enrichment of SrcDEL10 from the library by binding of the phosphorylated molecule to the 4G10 antibody and with the activity observed in the ADP-Glo assay being from a phosphotransfer to the from the library by binding of the phosphorylated molecule to the 4G10 antibody and with the activity small molecule. observed in the ADP-Glo assay being from a phosphotransfer to the small molecule.

Figure 4. Synthesized derivatives of SrcDEL10.

We then questioned if SrcDEL10 was indeed selected from the library based on the ability to serve as a Src substrate and accept a phosphate. Using purified DNA conjugates to SrcDEL10, 10–6, and 10–8, we confirmed that the phenolic oxygen was, in fact, required for enrichment in a qPCR selection assay and that the kinase treatment is also required for enrichment (Figure 5b). While the lower enrichment (100-fold) relative to LYNtide (10,000-fold) could be explained by differences in antibody affinity, the LC/MS assay would suggest this difference largely reflects the level of

phosphorylation. Figure 4. Synthesized derivatives of SrcDEL10. Figure 4. Synthesized derivatives of SrcDEL10.

We then questioned if SrcDEL10 was indeed selected from the library based on the ability to serve as a Src substrate and accept a phosphate. Using purified DNA conjugates to SrcDEL10, 10–6, and 10–8, we confirmed that the phenolic oxygen was, in fact, required for enrichment in a qPCR selection assay and that the kinase treatment is also required for enrichment (Figure 5b). While the lower enrichment (100-fold) relative to LYNtide (10,000-fold) could be explained by differences in antibody affinity, the LC/MS assay would suggest this difference largely reflects the level of phosphorylation.

Molecules 2019, 24, x FOR PEER REVIEW 9 of 19 (a)

(b) (a) Figure 5. Structure activity relationship (SAR) studies of SrcDEL10 derivatives. ( a) ADP-Glo assays of the SrcDEL10 derivative, SrcDEL10–6, lacking a phenolic oxygen show comparable activity to SrcDEL10.. Derivative Derivative SrcDEL10SrcDEL10––88,, which which contains contains the the phenol phenol but but lacks lacks the the cyclopropyl cyclopropyl group, group, shows shows no activity.activity. (b(b)) Analysis Analysis of of enrichment enrichment in thein the substrate substrate selection selection (shown (shown in Scheme in Scheme1) shows 1) theshows phenol the phenolis required is required for enrichment. for enrichment. (10-NE, (10-NE,SrcDEL10 SrcDEL10with no with enzyme no enzyme treatment). treatment).

2.4. SrcWe Activity then questioned Inhibition if SrcDEL10 was indeed selected from the library based on the ability to serve as a Src substrate and accept a phosphate. Using purified DNA conjugates to SrcDEL10, 10–6, and The original six off-DNA hits were tested for inhibition of c-Src activity. For these tests, we used 10–8, we confirmed that the phenolic oxygen was, in fact, required for enrichment in a qPCR selection 32P-ATP as a substrate and detected phosphorylation levels of a biotinylated-LYNtide substrate after assay and that the kinase treatment is also required for enrichment (Figure5b). While the lower adsorption to streptavidin coated membranes [16]. This assay should not be affected by activation of ATP hydrolysis or phosphotransfer to the small molecules. To avoid potential inhibition from reduction in ATP concentration, turnover levels were kept low (~1%), and ATP levels were confirmed to be negligibly reduced by ADP-Glo. Observed IC50 values ranged from 8.1 to 96 µM (Figures 6 and S10). These values suggest modest binding affinity (~low µM) of the compounds given the use of 50 µM biotin-LYNtide (Km = 5.2 µM) in these assays. Assay sensitivity was not sufficient for detection of such low turnover at significantly lower concentrations of biotin-LYNtide. For SrcDEL10, inhibition was additional validated by an HPLC assay of the LYNtide phosphorylation under similar conditions (Figure S11). There was no apparent correlation of these values to the Km or kcat values in the prior assays as substrates.

2.0 IC50 for SrcDEL10 IC50 1.5

Compound µM 1.0 0.5 % TURN OVER TURN % SrcDEL5 64 0.0 -2 0 2 Log (SrcDEL10 (μM)) SrcDEL8 96 10 IC for SrcDEL21 SrcDEL10 48 1.0 50 SrcDEL14 18 SrcDEL16 26 0.5

SrcDEL21 8.0 TURN% OVER 0.0 -2 0 2 Log (SrcDEL21 (μM)) 10

(a) (b)

Figure 6. Inhibition of hit molecules in a radiolabel assay with 32P-ATP (50 µM) and biotinylated-

LYNtide (50 µM) as substrates. (a) Half maximal inhibitory concentration (IC50) for six hit structures (structures shown in Table 1). (b) Representative data for SrcDEL10 and SrcDEL21.

2.5. NMR Analysis To gain insight in to the binding of SrcDEL10 to c-Src, we performed NMR spectroscopy to measure changes in chemical shifts in the presence of SrcDEL10. Such changes are due to either the ligand altering the magnetic environment of protein amide groups through direct interactions, which can potentially identify specific residues that interact with the ligand, or to effects of binding on the overall protein conformational equilibrium. We collected 1H,15N heteronuclear single quantum

Molecules 2019, 24, x FOR PEER REVIEW 9 of 19

(b)

Figure 5. Structure activity relationship (SAR) studies of SrcDEL10 derivatives. (a) ADP-Glo assays of the SrcDEL10 derivative, SrcDEL10–6, lacking a phenolic oxygen show comparable activity to MoleculesSrcDEL102019. Derivative, 24, 2764 SrcDEL10–8, which contains the phenol but lacks the cyclopropyl group, shows9 of 19 no activity. (b) Analysis of enrichment in the substrate selection (shown in Scheme 1) shows the phenol is required for enrichment. (10-NE, SrcDEL10 with no enzyme treatment). enrichment (100-fold) relative to LYNtide (10,000-fold) could be explained by differences in antibody affinity, the LC/MS assay would suggest this difference largely reflects the level of phosphorylation. 2.4. Src Activity Inhibition 2.4. Src Activity Inhibition The original six off-DNA hits were tested for inhibition of c-Src activity. For these tests, we used 32P-ATP asThe a originalsubstrate six and off-DNA detected hits werephosphorylation tested for inhibition levels ofof c-Srca biotinylated-LYNtide activity. For these tests, substrate we used after 32 adsorptionP-ATP to as streptavidin a substrate and coated detected membranes phosphorylation [16]. This levels assay of ashould biotinylated-LYNtide not be affected substrate by activation after of ATPadsorption hydrolysis to or streptavidin phosphotransfer coated membranes to the small [16]. molecules. This assay should To avoid not bepotential affected byinhibition activation from reductionof ATP in hydrolysis ATP concentration, or phosphotransfer turnover to levels the small were molecules.kept low (~1%), To avoid and potential ATP levels inhibition were confirmed from reduction in ATP concentration, turnover levels were kept low (~1%), and ATP levels were confirmed to be negligibly reduced by ADP-Glo. Observed IC50 values ranged from 8.1 to 96 µM (Figures 6 and to be negligibly reduced by ADP-Glo. Observed IC50 values ranged from 8.1 to 96 µM (Figure6 and S10).Figure These S10).values These suggest values modest suggest binding modest affinity binding (~low affinity µM) (~low of µtheM) compounds of the compounds given giventhe use the of 50 µM biotin-LYNtide (Km = 5.2 µM) in these assays. Assay sensitivity was not sufficient for detection use of 50 µM biotin-LYNtide (Km = 5.2 µM) in these assays. Assay sensitivity was not sufficient for of suchdetection low ofturnover such low turnoverat significantly at significantly lower lower concentrations concentrations of of biotin-LYNtide. biotin-LYNtide. For ForSrcDEL10 SrcDEL10, , inhibitioninhibition was was additional additional validate validatedd by by an an HPLC HPLC assay assay ofof thethe LYNtide LYNtide phosphorylation phosphorylation under under similar similar conditionsconditions (Figure (Figure S11). S11). There There was was no no appare apparentnt correlationcorrelation of of these these values values to theto the Km orKm k orcat valueskcat values in in the priorthe prior assays assays as substrates. as substrates.

2.0 IC50 for SrcDEL10 IC50 1.5

Compound µM 1.0 0.5 % TURN OVER TURN % SrcDEL5 64 0.0 -2 0 2 Log (SrcDEL10 (μM)) SrcDEL8 96 10 IC for SrcDEL21 SrcDEL10 48 1.0 50 SrcDEL14 18 SrcDEL16 26 0.5

SrcDEL21 8.0 TURN% OVER 0.0 -2 0 2 Log (SrcDEL21 (μM)) 10

(a) (b)

FigureFigure 6. Inhibition 6. Inhibition of of hit hit molecules in in a radiolabel a radiolabel assay assay with 32 withP-ATP 32 (50P-ATPµM) and(50 biotinylated-LYNtideµM) and biotinylated-

LYNtide(50 µ M)(50 as µM) substrates. as substrates. (a) Half ( maximala) Half maximal inhibitory inhibitory concentration concentration (IC50) for six hit(IC structures50) for six (structures hit structures (structuresshown inshown Table 1in). Table ( b) Representative 1). (b) Representative data for SrcDEL10 data forand SrcDEL10SrcDEL21 and. SrcDEL21. 2.5. NMR Analysis 2.5. NMR Analysis To gain insight in to the binding of SrcDEL10 to c-Src, we performed NMR spectroscopy to measure changesTo gain ininsight chemical in shiftsto the in binding the presence of SrcDEL10 of SrcDEL10 to .c-Src, Such changeswe performed are due toNMR either spectroscopy the ligand to measurealtering changes the magnetic in chemical environment shifts in of the protein presence amide of groups SrcDEL10 through. Such direct changes interactions, are due which to either can the ligandpotentially altering identifythe magnetic specific environment residues that interact of protei withn amide the ligand, groups or to through effects of direct binding interactions, on the overall which 1 15 can potentiallyprotein conformational identify specific equilibrium. residues We that collected interactH, withN heteronuclear the ligand, or single to effects quantum of binding coherence on the 15 overall( N-HSQC) protein spectraconformational of the phosphorylated equilibrium. c-Src:ATP We collected protein complex 1H,15N both heteronuclear in the presence single and absence quantum of SrcDEL10. Addition of the molecule produced chemical shift differences, or perturbations, as well as changes in peak intensity of resonances (Figure7a,b, Figure S12). A number of c-Src:ATP resonances have low intensity, and some, such as G395, were lost or highly attenuated in the presence of SrcDEL10. Mapping the chemical shifts with changes to the Src crystal structure shows that the residues are mostly in the large lobe (bottom lobe as viewed in Figure7c), but the changes are not su fficiently localized to one region to suggest a binding site. As protein kinases are well known to be conformationally flexible, it is not surprising that we observe changes in peak intensity and a broad spatial distribution of the affected residues, which likely reflects a more global conformational response to binding rather Molecules 2019, 24, x FOR PEER REVIEW 10 of 19 coherence (15N-HSQC) spectra of the phosphorylated c-Src:ATP protein complex both in the presence and absence of SrcDEL10. Addition of the molecule produced chemical shift differences, or perturbations, as well as changes in peak intensity of resonances (Figures 7a,b, S12). A number of c-Src:ATP resonances have low intensity, and some, such as G395, were lost or highly attenuated in the presence of SrcDEL10. Mapping the chemical shifts with changes to the Src crystal structure shows that the residues are mostly in the large lobe (bottom lobe as viewed in Figure 7c), but the changes are not sufficiently localized to one region to suggest a binding site. As protein kinases are well known to be conformationally flexible, it is not surprising that we observe changes in peak intensityMolecules and2019 ,a24 broad, 2764 spatial distribution of the affected residues, which likely reflects a more10 global of 19 conformational response to binding rather than direct interactions. Dispersed changes with ligand bindingthan direct to kinases interactions. have been Dispersed observed changes for many with ligand kinases, binding including to kinases protein have kinase been observedA, p38 kinase, for FGFR1,many and kinases, c-Src including [53–57]. proteinOur results kinase suggest A, p38 kinase,SrcDEL10 FGFR1, affects and c-Src [conformational53–57]. Our results equilibrium, suggest butSrcDEL10 the chemicalaffects shift c-Src perturbations conformational are equilibrium, small and but ligand the chemical association shift perturbations does not greatly are small alter and the magneticligand association environment does of not c-Src greatly amide alter thegroups magnetic at the environment binding site. of c-Src Interestingly, amide groups binding at the binding was not observedsite. Interestingly, in the absence binding of ATP. was not observed in the absence of ATP.

A C

B

15 FigureFigure 7. 7.NMRNMR analysis analysis of of SrcDEL1SrcDEL100 binding.binding. ( (aa)) Overlay Overlay ofof 15N-HSQC spectraspectra of of the the phosphorylated phosphorylated SrcSrc catalytic catalytic domaindomain in in the the presence presence of ATP of (green) ATP or(green) ATP and orSrcDEL10 ATP and(red). SrcDEL10 Ligand concentrations (red). Ligand are near saturation based on estimated Km values. A portion of the spectrum showing significant concentrations are near saturation based on estimated Km values. A portion of the spectrum showing changes is highlighted. See Figure SX for full spectrum. (b) A portion of the spectrum shows significant significant changes is highlighted. See Figure SX for full spectrum. (b) A portion of the spectrum chemical shift perturbation of K321 in the presence of SrcDEL10.(c) Chemical shifts with large changes shows significant chemical shift perturbation of K321 in the presence of SrcDEL10. (c) Chemical shifts upon addition of SrcDEL10 are mapped (red) to the ribbon drawing of the Src kinase domain (PDB ID: with large changes upon addition of SrcDEL10 are mapped (red) to the ribbon drawing of the Src 1Y57) [58]. kinase domain (PDB ID: 1Y57) [58]. 2.6. Activity in Cell Culture 2.6. Activity in Cell Culture We evaluated the ability of SrcDEL10 to inhibit the activation of Src-dependent kinase signaling in EGFR-transformedWe evaluated the mammary ability of epithelial SrcDEL10 (NME) to inhibit cells [the59]. activation Epidermal of growth Src-dependent factor (EGF) kinase stimulation signaling in ofEGFR-transformed NME cells leads to mammary STAT3 phosphorylation epithelial (NME) via a Src-dependentcells [59]. Epidermal process [60growth]. To improvefactor (EGF) the stimulationmolecular propertiesof NME cells for cellleads permeability, to STAT3 we phosph preparedorylation a methyl via ester a Src-dependent derivative of SrcDEL10 process [60].at the To improveaspartate the side molecular chain. Treatment properties of cells for withcell permeabi this derivativelity, atwe 100 preparedµM prior a to methyl EGF stimulation ester derivative showed of SrcDEL10a marked at reduction the aspartate in STAT3 side chain. phosphorylation Treatment levelsof cells (Figure with 8this). No derivative reduction atwas 100 observedµM prior withto EGF stimulationthe parent showedSrcDEL10 a marked(data not reduction shown). The in STAT3 broad specificity phosphorylation Src family levels inhibitor, (FigurePP2 8)., was No used reduction as a waspositive observed control. with Importantly, the parent phosphorylationSrcDEL10 (data ofnot ERK1 shown)./2, which Theoccurs broad viaspecificity a Src independent Src family pathway, inhibitor, PP2was, was not used reduced. as a positive Similarly, control. autophosphorylation Importantly, phosphorylation of EGFR at Y1068 of was ERK1/2, not aff whichected. occurs These resultsvia a Src suggest that SrcDEL10 is not a general protein tyrosine kinase inhibitor, but can specifically reduce independent pathway, was not reduced. Similarly, autophosphorylation of EGFR at Y1068 was not Src-mediated signaling events. affected. These results suggest that SrcDEL10 is not a general protein tyrosine kinase inhibitor, but can specifically reduce Src-mediated signaling events.

Molecules 2019, 24, 2764 11 of 19 Molecules 2019, 24, x FOR PEER REVIEW 11 of 19

DMSO PP2 ScrDEL 10-ester

hours 3 3 6 10 24 3 3 6 10 24 EGF - + - + + + + -+ + + +

pEGFR (Y1068)

tE G F R

pSTAT 3 (Y705) tSTAT3

pERK1/2

tERK1/2

Tubulin

FigureFigure 8. Inhibition 8. Inhibition of ofSrc-dependent Src-dependent kinase kinase signaling signaling in in EGFR-transformed EGFR-transformed mammary mammary epithelial epithelial cells cells (NME)(NME) cells cells by by SrcDEL10SrcDEL10. NME. NME cells cells were were serum serum deprived deprived forfor 2424 hh inin thethe presence presence of of vehicle vehicle control control (DMSO),(DMSO), Src Src family family inhibitor inhibitor PP2PP2 (10(10 µM),µM), or or SrcDEL10-esterSrcDEL10-ester (100(100µ µM)M) for for the the indicated indicated amounts amounts of of time.time. Where Where indicated, indicated, these these cells cells were were subsequently subsequently stimulated stimulated withwith epidermalepidermal growth growth factor factor (EGF) (EGF) (50(50 ng/mL) ng/mL) for for an an additional additional 30 30 min. min. Equal Equal protei proteinn aliquots werewere analyzedanalyzed by by immunoblot immunoblot with with the the indicated antibodies. Data are representative of at least 3 independent experiments. indicated antibodies. Data are representative of at least 3 independent experiments. 3. Summary Discussion and Conclusion 3. Summary Discussion and Conclusion We prepared a peptidomimetic DNA-encoded chemical library and applied a selection to enrich forWe substrates prepared to a the peptidomimetic tyrosine kinase DNA-encoded c-Src. Assessment chemical of six library hit molecules and applied as substrates a selection for c-Srcto enrich in foran substrates assay that to measures the tyrosine ADP kina productionse c-Src. (ADP-Glo)Assessment gave of six Km hitvalues molecules in the as mid-micromolar substrates for rangec-Src in an (13–33assay thatµM), measures which compares ADP production favorably to (ADP-Glo) the Km of an gave optimal Km values peptide in substrate the mid-micromolar LYNtide (Km range= 5 µM (13–). 33 SubsequentµM), which analysis compares indicated, favorably however, to the K thatm of the an observed optimal activitypeptidewas substrate largely LYNtide due to activation (Km = 5 µM). of SubsequentATP hydrolysis analysis and indicated, not phosphotransfer however, that for at the least ob 4served of the 6activity hits. Nonetheless, was largely all due six to hit activation molecules of ATPdisplayed hydrolysis inhibitory and not activity phosphotransfer in a radiolabel for assay at least with 4 ICof 50thevalues 6 hits. ranging Nonetheless, from 8-100 all sixµM. hit The molecules ability displayedto serve inhibitory as a substrate activity was in confirmed a radiolabel for assay a selected with hit,IC50SrcDEL10 values ranging, by LC from/MS. 8-100 Application µM. The of ability the to selectionserve as assaya substrate using DNA-linked was confirmedSrcDEL10 for a indicatedselected thathit, enrichmentSrcDEL10, fromby LC/MS. the library Application was likely of due the selectionto the ability assay tousing serve DNA-linked as a substrate, SrcDEL10 as enrichment indicated was dependent that enrichment on both thefrom phenol the library oxygen was and likely the dueenzyme to the treatment.ability to Analysisserve as ofa substrate,SrcDEL10 aswith enrichment c-Src and ATPwas bydependent NMR indicated on both binding, the phenol and an oxygen ester andderivative the enzyme demonstrated treatment. inhibition Analysis of of Src SrcDEL10 signaling with in NME c-Src cells. and ATP by NMR indicated binding, The ATPase activity of protein kinases is well documented and is commonly observed background and an ester derivative demonstrated inhibition of Src signaling in NME cells. signal in many kinase assay approaches. Compared to phosphotransfer to an optimal peptide The ATPase activity of protein kinases is well documented and is commonly observed substrate, ATPase activity is generally dramatically lower (~two to three orders of magnitude) [61]. background signal in many kinase assay approaches. Compared to phosphotransfer to an optimal Here, the ATPase activity of c-Src (based on kcat/Km) was activated to within 10-fold of the LYNtide peptide substrate, ATPase activity is generally dramatically lower (~two to three orders of phosphotransfer activity. This is well above background levels of ATPase activity of Src alone, which magnitude) [61]. Here, the ATPase activity of c-Src (based on kcat/Km) was activated to within 10-fold were below the limit of detection under our assay conditions. Prior work has shown that ATPase of activitythe LYNtide can be phosphotransfer regulated in parallel activi withty. This protein is well kinase above activity background [62]. We hypothesize levels of ATPase that SrcDEL10 activity of Srcactivates alone, which ATPase were activity below by the binding limit of to detection and stabilizing under theour activeassay formconditions. of c-Src. Prior The work substrate has shown and thatATP ATPase hydrolysis activity activation can be capabilities regulated mayin parallel be connected, with protein given activationkinase activity was observed [62]. We with hypothesize several thatof SrcDEL10 the structurally activates diverse ATPase hits that activity were by selected binding on theto and ability stabilizing to accept the a phosphate. active form Also, of c-Src. SAR ofThe substrateSrcDEL10 andderivatives ATP hydrolysis indicated activation that the ATPase capabilities activation may is likelybe connected, a specific interaction. given activation While this was observedmay be anwith uncommon several of phenomenon, the structurally it is adiverse cautionary hits note that as were a potential selected source on the of assayability interference to accept a phosphate.for the study Also, of SAR kinase of inhibitorsSrcDEL10 and derivatives activators. indicated that the ATPase activation is likely a specific interaction.In our While library this selection, may be selection an uncommon stringency phenom was fairlyenon, low it is with a cautionary a relatively note high as amount a potential of sourceenzyme of assay used, interference which may befor responsible the study of for kinase the modest inhibitors ability and of theactivators. hit SrcDEL10 to serve as a true substrate.In our library Setting selection, the stringency selection of a selection stringency appropriately was fairly may low depend with a onrelatively the desired high outcome amount of of enzyme used, which may be responsible for the modest ability of the hit SrcDEL10 to serve as a true substrate. Setting the stringency of a selection appropriately may depend on the desired outcome of developing a substrate competitive inhibitor versus developing more active substrates. For inhibitors, there is no need to select for molecules that have high kcat values. Based on prior work with c-Src, we presumed that it would be difficult for small molecules to serve as highly active substrates compared

Molecules 2019, 24, 2764 12 of 19 developing a substrate competitive inhibitor versus developing more active substrates. For inhibitors, there is no need to select for molecules that have high kcat values. Based on prior work with c-Src, we presumed that it would be difficult for small molecules to serve as highly active substrates compared to larger (typically at least 9-mer) peptide substrates [24]. Further work may explore libraries which contain larger molecular weight molecules with increasing peptide character. For development of more active substrates, conducting multiple selections with titration of the enzyme down to low levels may be more appropriate. Also, combining both substrate and affinity selections may be a useful strategy. Part of the rationale for employing a substrate-mediated selection was the potential for sensitivity in enriching low affinity hits due to enzymatic turnover. High specific activity, which is a critical requirement for affinity-mediated selections, can be problematic for protein kinases. Our attempts at affinity-based selection with an immobilized c-Src were unsuccessful for either a DNA-linked LYNtide or SrcDEL10, which we presume was due to low specific activity of the enzyme as selections with ligands of low micromolar affinity are generally successful [63]. Given the applicability of substrate-mediated selections in both phage display and mRNA display [8,9], such selections should find additional application with DELs for both discovery of substrate competitive inhibitors and improved activity probes. Prior work from our lab has demonstrated DEL-compatible serine/threonine kinase, protease, and transferase substrate-mediated selections for activity detection using DNA-linked substrates [7]. We hope that this study will inspire others to apply these methods to DELs for discovery of new substrate molecules.

4. Materials and Methods

4.1. DNA Library Preparation Following previously published methods [10], individual 40-mer oligonucleotides (Bioneer) containing 20 base common sequences and 20 bases of a unique variable region were treated with T4 polynucleotide kinase (PNK). Orthogonal 20 bases sequences for library assembly used were as previously reported [7,10]. Following, the 50-phosphorylated oligos were combined based on complementarity in two pools so that Pool A contained variable regions 1–48 and Pool B contained variable regions 49–96 and then ligated by T4 DNA ligase. Following ligation, the pools were separately purified by PAGE, collected by electroelution, and then ethanol precipitated. DNA was quantified by UV absorbance and qPCR. Using approximately 10,000 copies of each library member as the template, each pool (variable sequences in each pool: Pool A = 1–48, Pool B = 49–96) was PCR amplified separately using with 0.5 µM end primers (one end primer contained the T7 promoter sequence), 1X DreamTaq buffer, 0.2 mM dNTPs, and 0.025 U/µL DreamTaq DNA polymerase for 20 cycles. The crude PCR products combined and by SPRI clean-up [64]. This was repeated using fresh aliquots of template until 1 nmol of each library was collected, as determined by UV and confirmed by qPCR using common end primers. Each pool of dsDNA was transcribed 1X Thermo Scientific transcription buffer, 5 mM NTPs, 20 mM additional MgCl2, 200 nM dsDNA template (as determined by qPCR using T7-promoter primer), with 3 U/µL T7 RNA polymerase and incubated at 37 ◦C for 16 h. Upon incubation, a white precipitate was observed and 10 U DNase I was added and incubated at 37 ◦C for 30 min, followed by the addition of 1.5 eq. (relative to total Mg2+) of EDTA. RNA was purified using PureLink RNA Mini kits (Thermo Fisher) and quantified by UV absorbance. Each pool of RNA at 12 µM (final concentration) was combined with 1X First Strand buffer (Invitrogen), 10 mM DTT, 1.0 mM dNTPs, 1.2 eq. (relative to RNA) of 50-amine modified forward primer, and 1.0 U/µL SuperScript IV reverse polymerase. First, the RNA, dNTPs, and primer were incubated at 65 ◦C for 5 min and then placed on ice for at least 5 min. To this mixture, the buffer, DTT, and enzyme are added. The combined mixture was incubated at 47 ◦C for 16 h and purified as the RNA/DNA heteroduplex by SPRI clean-up. Total DNA was quantified by qPCR after single cycle PCR (primer extension). Molecules 2019, 24, 2764 13 of 19

4.2. Chemical Translation

Pools of A and B library RNA/DNA heteroduplex were hydrolyzed in 0.1 M NaCO3 buffer, pH 10.3 and heated to 95 ◦C for 5 min, then 1.5 eq. of all cognate common primers were added and the mixture was slowly cooled to RT. Following, the resulting ssDNA was taken up in 1X hybridization buffer (2X SSC, 10 mM Tris, pH 8, 1 mM EDTA, pH 8, 0.005% (v/v) Triton X-100, 0.02% (w/v) SDS, 0.5 mg/mL BSA, 0.5 mg/mL sheared salmon sperm DNA) to a final volume of 15 mL. Meanwhile, the array was pre-blocked with 1X hybridization buffer for 15 min at 37 ◦C, solution decanted and the ssDNA-containing hybridization solution was added to the array, and incubated at 37 ◦C for 36 h with agitation. Following hybridization, the solution was decanted from the array, and the array was washed 2 25 mL 1X hybridization buffer, 1 25 mL 10 SSC, and 1 25 mL 0.25 SSC. The array × × × × × was then assembled into the transfer apparatus and hybridized DNA was eluted with 4 35 µL array × elution buffer (10 mM NaOH, 1 mM EDTA, 0.005% Triton X-100), prepared fresh, and collected into a 384-well plate. Each elution was transferred to 20 µL DEAE Sepharose on a 384-well filter plate with 16 µL 60 mM HOAc and incubated for 5 min at RT. The second array elution was added to the DEAE-containing filter plate with an additional 16 µL 60 mM HOAc and incubated again for 5 min. The flow through was collected by centrifugation and transfer was repeated for the final two array elutions. The DNA-loaded filter plate was then washed 1 80 µL DEAE bind buffer, then 3 80 µL MeOH. × × Building blocks added are shown in Table S1. Briefly, Fmoc-amino acids were coupled by adding 50 µL of 50 mM Fmoc amino acids, 50 mM EDC-HCl, 5 mM HOAt in 40% DMF/60% MeOH to the filter plate and incubated for 30 min at RT, similar to previous reports [38]. The reaction mixture was drained from the 384-well filter plate and a fresh reaction mixture was prepared as described above and incubated again for 30 min at RT. The filter plate was then washed 3 80 µL MeOH followed by 3 80 × × µL DMF. Following, 50 µL 20% (v/v) piperidine in DMF was added to each well following coupling of an Fmoc-amino acid and incubated for 30 min at RT. Each well was then washed 3 80 µL DMF × followed by 3 80 µL MeOH. For peptoid monomers, chloroacetate was coupled by adding 50 µL of × 100 mM sodium chloroacetate and 150 mM DMTMM-Cl in MeOH for 30 min at RT, with a repeated coupling using a fresh reaction mixture, as previously reported [39]. Following, resin was washed 3 × 80 µL MeOH and then 3 80 µL DMSO. For displacement, 50 µL of a 2 M stock of the desired primary × amine in DMSO was added and incubated for 16 h at RT, followed by washing 6 80 µL DMSO, × 3 80 µL MeOH, and 80 µL DEAE bind buffer. For benzyl-based peptoids, a solution of 100 mM × 4-(chloromethyl)benzoic acid with 150 mM DIEA and 100 mM DMTMM-BF4 in DMF was prepared, added to the desired wells, and incubated at RT for 30 min. Coupling was repeated, and followed by washing with 3 80 µL DMF, 3 80 µL MeOH, 3 80 µL DMSO. Displacement was achieved × × × followed the same procedure as with chloroacetate peptoids. Prior to elution from DEAE Sepharose, all wells were washed 3 80 µL DMF, 3 80 µL MeOH, × × and lastly 80 µL DEAE bind buffer. For elution, 35 µL of DPCC DEAE elution buffer (10 mM NaOH, 1.5 M NaCl, 0.005% Triton X-100) was added and incubated for 5 min at RT. The elution was collected by centrifugation into a 384-well plate and elution was repeated for a total of four times. The collected elutions were pooled in a 50 mL 10 kDa MWCO centrifugal filter, neutralized with 1.0 eq. HOAc (relative to total NaOH) and Tris, pH 8.0 was added to a final concentration of 10 mM. The solution was then concentrated by centrifugation and washed 3 TE. The recovery of translated library was × quantified using qPCR. Chemical translation was continued following the same steps as described by hybridization to Array C (step 1), Array A (step 2), and Array D (step 3), yielding the 3-mer DPCC library. Cartridge split for step 4. Following the completion of step 3, the 3-mer DPCC library was taken in 1X hybridization buffer (2X SSC, 10 mM Tris, pH 8, 1 mM EDTA, pH 8, 0.005% (v/v) Triton X-100, 0.02% (w/v) SDS, 0.5 mg/mL BSA, 0.5 mg/mL sheared salmon sperm DNA) to a final volume of 3.75 mL and then split across 5 cartridges (attached in series) containing variable sequences (denoted F50-F90) by incubation for 16 h at 37 ◦C with constant circulation by a peristaltic pump. The hybridization solution was collected and the cartridges were washed by 50 mL 1X hybridization buffer, 25 mL 10X SSC, and 25 mL Molecules 2019, 24, 2764 14 of 19

0.25X SSC. The cartridges were then removed and separately eluted with 2 mL array elution buffer and neutralized with HOAc. Following quantification of the fully translated library by qPCR, the DNA was suspended in 1X DreamTaq buffer with 0.2 mM dNTPs, 1 µM reverse end primer, and 0.05 U/µL DreamTaq DNA polymerase for a single cycle PCR with a 20-min extension time to convert the translated ssDNA to dsDNA. The dsDNA was purified by SPRI clean-up. The translated dsDNA was deprotected by suspending in 250 mM sodium borate, pH 9.4 and then incubating for 2 h at 80 ◦C[65], followed by purification by SPRI cleanup and quantification by qPCR.

4.3. Substrate-Mediated Selection for Tyrosine Kinases For optimization of substrate-based selection, two unique 180-mer constructs were prepared by PCR, as previously described [7]. Each contained two unique 20-mer sequences for specific amplification and detection in qPCR. One was prepared with an unmodified forward primer as a control. The other was prepared using a LYNtide 20-mer oligo conjugate as a primer. For the selection of the library, 9 fmol of the deprotected translated dsDNA library was mixed with 500 zmol of a control LYNtide construct containing unique barcodes. This mixture was treated with recombinant c-Src kinase catalytic domain at approximately 2 µM concentration overnight at 30 ◦C. After kinase treatment, the mixture was phenol/chloroform extracted and precipitated from ethanol. 1.6 µL Protein G magnetic beads (ThermoFisher) were incubated with 10 µg anti-phosphotyrosine IgG. The antibody-loaded magnetic beads were washed with PBST with 1 mg/mL BSA, 1 mg/mL tRNAs) to remove any non-specifically bound antibody. Following, the precipitated kinase-treated library was taken up in 20 µL of the above buffer and incubated with the antibody-loaded magnetic beads. The magnetic beads were then washed 3 20 µL with the above buffer and 3 20 µL PBST. Captured library × × members were eluted by the addition of 20 µL of 1 mM phenylphosphate. The resulting supernatant was then used as a template in a PCR amplification with Illumina adaptor and indexes sequences, as previously described [7], and purified products submitted for analysis by Illumina MiSeq. Barcodes were assigned to codon numbers, as previously described [66], and enrichment calculated relative to the unselected library.

4.4. qPCR Assay Kinase reaction was performed as follows 1x kinase buffer (25 mM Tris-HCl (pH 7.5), 2 mM dithiothreitol (DTT), 0.1 mM Na3VO4, 10 mM MgCl2), 1 mM ATP, different concentrations (1 µM to 100 µM) of substrates (either off DNA or on DNA hit molecules), and 12 nM rSrc by incubation for 2 h at 30 ◦C. After that, distinct 55mers were annealed to each kinase reaction, as previously described [67]. The annealed reactions were incubated with anti Pho-Tyr loaded protein G magnetic bead at RT for 30 min. After washing by three times 20 µL 1xPBST with 1 mg/mL BSA, 1 mg/mL tRNAs), the captured samples were eluted by 20 µL of 1 mM phenylphosphate. The resulting supernatant was then used as a template in qPCR.

4.5. ADP-Glo Assay After the kinase reaction was performed as previous section, the reaction mixture (5 µL) was incubated with 5 µL of ADP-Glo™ reagent at RT for 40 min and then add 10 µL of kinase detection reagent at RT for 1 h. The luminescence was measure by plate reader.

4.6. NADH Coupled Assay Kinase reaction was performed as follows 1x kinase buffer (25 mM Tris-HCl (pH 7.5), 2 mM dithiothreitol (DTT), 0.1 mM Na3VO4, 10 mM MgCl2), 1 mM ATP, 100 µM peptides and 12 nM Src at 30 ◦C for 3 h. The kinase reaction mixture was incubated with 30 unit/mL lactic dehydrogenase (LDH), 20 unit/mL pyruvate kinase (PK), 2 mM phosphoenolpyruvate (PEP), and 1 mM NADH. The absorbance of NADH was monitored at 340 nm. Molecules 2019, 24, 2764 15 of 19

4.7. Radiolabeled Assay

Each hit molecule was preincubated with 1.2 nM c-Src at 30 ◦C for 30 min. The kinase reaction was initiated with the addition of 50 µM LYNtide and 50 µM ATP with 0.15 µCi gamma 32P ATP at 30 ◦C for 10 min. The kinase reaction samples (2 µL) were spotted on P81 filter paper consisting of 1.5 cm squares. After air dry, the P81 papers were washed 4 times with 0.5% phosphoric acid for 5 min each. The P81 papers were washed one time with acetone for 5 min. The papers were then allowed to air dry for about 20 min. The dried P81 papers were analyzed by liquid scintillation counter.

4.8. Cell Signaling Assays NME cells were constructed via stable overexpression of EGFR in normal murine mammary gland (NMuMG) cells as previously described [60]. These cells were cultured in DMEM containing 10% FBS, 10 µg/mL insulin, penicillin, and streptomycin. For cell signaling assays, cells were plated for 24 h prior to removal of the FBS for an additional 24 h. During this serum starvation, PP2 (Sigma, 529576) or SrcDEL10-ester were added as indicated, and cells were subsequently stimulated with EGF (50 ng/mL) (Goldbio, 1150-04) for 30 min. After this stimulation cells were lysed in a modified RIPA buffer containing 50 mM Tris, 150 mM NaCl, 0.25% Sodium Deoxycholate, 1.0% NP40, 0.1% SDS, protease inhibitor cocktail, 10 mM activated sodium ortho-vanadate, 40 mM β-glycerolphosphate, and 20mM sodium fluoride. Equal aliquots of these lysates were separated by reducing SDS PAGE and transferred to PVDF membranes. These membranes were probed with antibodies against phospho-EGFR Y1068, total EGFR, phosho-STAT3-Y705, total STAT3, phospho-ERK1/2, total ERK1/2 (Cell Signaling Technologies), and tubulin (DSHB). Differential binding of these antibodies was detected using their appropriate secondary antibodies.

4.9. Expression, Purification, and Phosphorylation of c-Src Catalytic Domain The wild type c-Src catalytic domain (residues 255–533) was co-expressed with YoPH phosphatase 15 in E. coli BL21 (DE3) cells and grow in M9 media containing NH4Cl. Uniformly labelled c-Src catalytic domain was purified by affinity and ion exchange chromatography as previously described [68]. To produce phosphorylated c-Src catalytic domain, purified protein was incubated in buffer containing 10-fold excess ATP, 20 mM MgCl2, 50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 5% glycerol, and 1 mM DTT for 1.5 h at room temperature. ATP was washed out by exhaustive buffer exchange (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 5 mM DTT) for NMR samples.

4.10. NMR Experiments NMR samples of 15N-labeled phosphorylated c-Src kinase domain were concentrated with a stirred concentrator. Final concentrations were phosphorylated [c-Src] = 200 µM, and [ATP] = 400 µM in the absence of SrcDEL10; and phosphorylated [c-Src] = 200 µM, [ATP] = 400 µM and [SrcDEL10] = 2 mM. The 15N TROSY-HSQC spectra were collected on an Avance Bruker 800 MHz spectrometer at 298K with 32 scans and 128 increments in the indirect 15N dimension.

Supplementary Materials: The following are available online, Figure S1: Codon and gene abundances in the DNA-encoded library. Figure S2: Cubic plot analysis of a quality control DEL selection. Figure S3: Test enrichment assay with a LYNtide-DNA conjugate. Figure S4: Cubic plot analysis of DEL selection with B library. Figure S5: 24 hit molecules synthesized on-DNA for hit validation. Figure S6: qPCR assessed enrichment of initial 24 ligands on-DNA. Figure S7: HPLC analysis of LYNtide Phosphorylation. Figure S8: NADH coupled assay of hit molecules with c-Src. Figure S9: LC/MS analysis of SrcDEL10 phosphorylation. Figure S10: IC50 curves for selected hits in the 32-P-ATP inhibition assay. Figure S11: HPLC-assessed inhibition of LYNtide phosphorylation by SrcDEL10. Figure S12: Full HSQC NMR spectrum for SrcDEL10 binding to c-Src:ATP. Table S1: Library monomer set for the first 3 synthetic cycles. Table S2: Library monomer set for synthetic cycle 4. Table S3: ADP-glo assay of SrcDEL10 derivatives. Supplementary methods and compound characterization. Molecules 2019, 24, 2764 16 of 19

Author Contributions: Library synthesis: K.E.D.; Selection design, optimization, and performance: D.K.; DNA sequence data processing: K.E.D. and C.J.K.; qPCR selection assays and Hit validation: D.K.; on and off-DNA hit resynthesis: Y.S.; NMR structure analysis: D.X. and C.B.P.; cell culture and Western Blot assays: H.C., H.L., and M.K.W.; conceptualization and project administration: C.J.K.; writing—original draft preparation: C.J.K.; writing—review and editing: D.K., Y.S., M.K.W., C.B.P., C.J.K.; supervision: M.K.W., C.B.P., and C.J.K.; funding acquisition: M.K.W., C.B.P., and C.J.K. Funding: This research was funded by the NIH (R35GM128894) to CJK, the NIH (R01GM039478) to CBP, and the American Cancer Society (RSG-16-172-01) to MKW. The authors gratefully acknowledge the support for Purdue University Mass Spectrometry and Genome Sequencing Shared Resources from the Purdue Center for Cancer Research, NIH grant P30 CA023168. Acknowledgments: The authors gratefully acknowledge Rachael R. Jetson for assistance in naïve DNA library preparation. Conflicts of Interest: The authors declare no conflict of interest.

References

1. Goodnow, R.A.; Dumelin, C.E.; Keefe, A.D. DNA-encoded chemistry: Enabling the deeper sampling of chemical space. Nat. Rev. Drug Discov. 2016.[CrossRef] 2. Neri, D.; Lerner, R.A. DNA-Encoded Chemical Libraries: A Selection System Based on Endowing Organic Compounds with Amplifiable Information. Annu. Rev. Biochem. 2018, 87, 479–502. [CrossRef] 3. Zimmermann, G.; Neri, D. DNA-encoded chemical libraries: Foundations and applications in lead discovery. Drug Discov. Today 2016, 21, 1828–1834. [CrossRef] 4. Decurtins, W.; Wichert, M.; Franzini, R.M.; Buller, F.; Stravs, M.A.; Zhang, Y.; Neri, D.; Scheuermann, J. Automated screening for small organic ligands using DNA-encoded chemical libraries. Nat. Protoc. 2016, 11, 764–780. [CrossRef] 5. Cuozzo, J.W.; Centrella, P.A.; Gikunju, D.; Habeshian, S.; Hupp, C.D.; Keefe, A.D.; Sigel, E.A.; Soutter, H.H.; Thomson, H.A.; Zhang, Y.; et al. Discovery of a Potent BTK Inhibitor with a Novel Binding Mode by Using Parallel Selections with a DNA-Encoded Chemical Library. ChemBioChem 2017, 18, 864–871. [CrossRef] 6. Cochrane, W.G.; Malone, M.L.; Dang, V.Q.; Cavett, V.; Satz, A.L.; Paegel, B.M. Activity-Based DNA-Encoded Library Screening. ACS Comb. Sci. 2019, 21, 425–435. [CrossRef] 7. Jetson, R.R.; Krusemark, C.J. Sensing Enzymatic Activity by Exposure and Selection of DNA-Encoded Probes. Angew. Chem. Int. Ed. Engl. 2016, 55, 9562–9566. [CrossRef] 8. Ratnikov, B.; Cieplak, P.; Smith, J.W. High throughput substrate phage display for protease profiling. Methods Mol. Biol. 2009, 539, 93–114. 9. Valencia, C.A.; Cotten, S.W.; Dong, B.; Liu, R. mRNA-display-based selections for proteins with desired functions: A protease-substrate case study. Biotechnol. Prog. 2008, 24, 561–569. [CrossRef] 10. Krusemark, C.J.; Tilmans, N.P.; Brown, P.O.; Harbury, P.B. Directed Chemical Evolution with an Outsized Genetic Code. PLoS ONE 2016, 11, 1–16. [CrossRef] 11. Barouch-Bentov, R.; Sauer, K. Mechanisms of Drug-Resistance in Kinases. Expert Opin. Investig. Drugs 2011, 20, 153–208. [CrossRef][PubMed] 12. Bhullar, K.S.; Lagarón, N.O.; McGowan, E.M.; Parmar, I.; Jha, A.; Hubbard, B.P.; Rupasinghe, H.P.V. Kinase-targeted cancer therapies: Progress, challenges and future directions. Mol. Cancer 2018, 17, 48. [CrossRef][PubMed] 13. Davis, M.I.; Hunt, J.P.; Herrgard, S.; Ciceri, P.; Wodicka, L.M.; Pallares, G.; Hocker, M.; Treiber, D.K.; Zarrinkar, P.P. Comprehensive analysis of kinase inhibitor selectivity. Nat. Biotechnol. 2011, 29, 1046–1051. [CrossRef][PubMed] 14. Scapin, G. Protein kinase inhibition: Different approaches to selective inhibitor design. Curr. Drug Targets 2006, 7, 1443–1454. [CrossRef][PubMed] 15. Breen, M.E.; Soellner, M.B. Small molecule substrate phosphorylation site inhibitors of protein kinases: Approaches and challenges. ACS Chem. Biol. 2015, 10, 175–189. [CrossRef] 16. Turk, B.E.; Hutti, J.E.; Cantley, L.C. Determining protein kinase substrate specificity by parallel solution-phase assay of large numbers of peptide substrates. Nat. Protoc. 2006, 1, 375–379. [CrossRef] 17. Brooijmans, N.; Chang, Y.-W.; Mobilio, D.; Denny, R.A.; Humblet, C. An enriched structural kinase database to enable kinome-wide structure-based analyses and drug discovery. Protein Sci. 2010, 19, 763–774. [CrossRef] Molecules 2019, 24, 2764 17 of 19

18. Deng, Y.; Alicea-Velazquez, N.L.; Bannwarth, L.; Lehtonen, S.I.; Boggon, T.J.; Cheng, H.-C.; Hytonen, V.P.; Turk, B.E. Global analysis of human nonreceptor tyrosine kinase specificity using high-density peptide microarrays. J. Proteome Res. 2014, 13, 4339–4346. [CrossRef] 19. Al-Obeidi, F.A.; Wu, J.J.; Lam, K.S. Protein tyrosine kinases: Structure, substrate specificity, and drug discovery. Pept. Sci. 1998, 47, 197–223. [CrossRef] 20. Lou, Q.; Leftwich, M.E.; McKay, R.T.; Salmon, S.E.; Rychetsky, L.; Lam, K.S. Potent Pseudosubstrate-based Peptide Inhibitors for p60c-src Protein Tyrosine Kinase. Cancer Res. 1997, 57, 1877–1881. 21. Alfaro-Lopez, J.; Yuan, W.; Phan, B.C.; Kamath, J.; Lou, Q.; Lam, K.S.; Hruby, V.J. Discovery of a Novel Series of Potent and Selective Substrate-Based Inhibitors of p60c-src Protein Tyrosine Kinase: Conformational and Topographical Constraints in Peptide Design. J. Med. Chem. 1998, 41, 2252–2260. [CrossRef][PubMed] 22. Bogoyevitch, M.A.; Barr, R.K.; Ketterman, A.J. Peptide inhibitors of protein kinases-discovery, characterisation and use. Biochim. Biophys. Acta 2005, 1754, 79–99. [CrossRef][PubMed] 23. Lee, J.H.; Nandy, S.K.; Lawrence, D.S. A highly potent and selective PKCalpha inhibitor generated via combinatorial modification of a peptide scaffold. J. Am. Chem. Soc. 2004, 126, 3394–3395. [CrossRef] [PubMed] 24. Breen, M.E.; Steffey, M.E.; Lachacz, E.J.; Kwarcinski, F.E.; Fox, C.C.; Soellner, M.B. Substrate activity screening with kinases: Discovery of small-molecule substrate-competitive c-Src inhibitors. Angew. Chem. Int. Ed. Engl. 2014, 53, 7010–7013. [CrossRef][PubMed] 25. Georghiou, G.; Kleiner, R.E.; Pulkoski-Gross, M.; Liu, D.R.; Seeliger, M.A. Highly specific, bisubstrate-competitive Src inhibitors from DNA-templated macrocycles. Nat. Chem. Biol. 2012, 8, 366. [CrossRef][PubMed] 26. Smith, G.K.; Wood, E.R. Cell-based assays for kinase drug discovery. Drug Discov. Today. Technol. 2010, 7, e1–e94. [CrossRef] 27. Folkvord, S.; Flatmark, K.; Dueland, S.; de Wijn, R.; Groholt, K.K.; Hole, K.H.; Nesland, J.M.; Ruijtenbeek, R.; Boender, P.J.; Johansen, M.; et al. Prediction of response to preoperative chemoradiotherapy in rectal cancer by multiplex kinase activity profiling. Int. J. Radiat. Oncol. Biol. Phys. 2010, 78, 555–562. [CrossRef] 28. Reimer, U.; Reineke, U.; Schneider-Mergener, J. Peptide arrays: From macro to micro. Curr. Opin. Biotechnol. 2002, 13, 315–320. [CrossRef] 29. Placzek, E.A.; Plebanek, M.P.; Lipchik, A.M.; Kidd, S.R.; Parker, L.L. A peptide biosensor for detecting intracellular Abl kinase activity using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Anal. Biochem. 2010, 397, 73–78. [CrossRef] 30. Peterson, L.B.; Imperiali, B. Real-Time and Continuous Sensors of Protein Kinase Activity Utilizing Chelation-Enhanced Fluorescence. Concepts Case Stud. Chem. Biol. 2014, 1–16. 31. Turner, A.H.; Lebhar, M.S.; Proctor, A.; Wang, Q.; Lawrence, D.S.; Allbritton, N.L. Rational Design of a Dephosphorylation-Resistant Reporter Enables Single-Cell Measurement of Tyrosine Kinase Activity. ACS Chem. Biol. 2016, 11, 355–362. [CrossRef][PubMed] 32. Bantscheff, M.; Eberhard, D.; Abraham, Y.; Bastuck, S.; Boesche, M.; Hobson, S.; Mathieson, T.; Perrin, J.; Raida, M.; Rau, C.; et al. Quantitative chemical proteomics reveals mechanisms of action of clinical ABL kinase inhibitors. Nat. Biotechnol. 2007, 25, 1035–1044. [CrossRef][PubMed] 33. Patricelli, M.P.; Szardenings, A.K.; Liyanage, M.; Nomanbhoy, T.K.; Wu, M.; Weissig, H.; Aban, A.; Chun, D.; Tanner, S.; Kozarich, J.W. Functional interrogation of the kinome using nucleotide acyl phosphates. Biochemistry 2007, 46, 350–358. [CrossRef][PubMed] 34. Halpin, D.R.; Harbury, P.B. DNA display I. Sequence-encoded routing of DNA populations. PLoS Biol. 2004, 2, E173. [CrossRef][PubMed] 35. Halpin, D.R.; Harbury, P.B. DNA display II. Genetic manipulation of combinatorial chemistry libraries for small-molecule evolution. PLoS Biol. 2004, 2, E174. [CrossRef] 36. Simon, R.J.; Kania, R.S.; Zuckermann, R.N.; Huebner, V.D.; Jewell, D.A.; Banville, S.; Ng, S.; Wang, L.; Rosenberg, S.; Marlowe, C.K. Peptoids: A modular approach to drug discovery. Proc. Natl. Acad. Sci. USA 1992, 89, 9367–9371. [CrossRef][PubMed] 37. Combs, D.J.; Lokey, R.S. Extended peptoids: A new class of oligomers based on aromatic building blocks. Tetrahedron Lett. 2007, 48, 2679–2682. [CrossRef] 38. Halpin, D.R.; Lee, J.A.; Wrenn, S.J.; Harbury, P.B. DNA display III. Solid-phase organic synthesis on unprotected DNA. PLoS Biol. 2004, 2, E175. [CrossRef] Molecules 2019, 24, 2764 18 of 19

39. Wrenn, S.J.; Weisinger, R.M.; Halpin, D.R.; Harbury, P.B. Synthetic ligands discovered by in vitro selection. J. Am. Chem. Soc. 2007, 129, 13137–13143. [CrossRef] 40. Schwochert, J.; Turner, R.; Thang, M.; Berkeley, R.F.; Ponkey, A.R.; Rodriguez, K.M.; Leung, S.S.F.; Khunte, B.; Goetz, G.; Limberakis, C.; et al. Peptide to Peptoid Substitutions Increase Cell Permeability in Cyclic Hexapeptides. Org. Lett. 2015, 17, 2928–2931. [CrossRef] 41. Tan, N.C.; Yu, P.; Kwon, Y.-U.; Kodadek, T. High-throughput evaluation of relative cell permeability between peptoids and peptides. Bioorg. Med. Chem. 2008, 16, 5853–5861. [CrossRef][PubMed] 42. Akritopoulou-Zanze, I.; Hajduk, P.J. Kinase-targeted libraries: The design and synthesis of novel, potent, and selective kinase inhibitors. Drug Discov. Today 2009, 14, 291–297. [CrossRef][PubMed] 43. Mincione, F.; Starnotti, M.; Menabuoni, L.; Scozzafava, A.; Casini, A.; Supuran, C.T. Carbonic anhydrase inhibitors: 4-sulfamoyl-benzenecarboxamides and 4-chloro-3-sulfamoyl-benzenecarboxamides with strong topical antiglaucoma properties. Bioorg. Med. Chem. Lett. 2001, 11, 1787–1791. [CrossRef] 44. Tinti, M.; Nardozza, A.P.; Ferrari, E.; Sacco, F.; Corallino, S.; Castagnoli, L.; Cesareni, G. The 4G10, pY20 and p-TYR-100 antibody specificity: Profiling by peptide microarrays. N. Biotechnol. 2012, 29, 571–577. [CrossRef] [PubMed] 45. Schmitz, R.; Baumann, G.; Gram, H. Catalytic specificity of phosphotyrosine kinases Blk, Lyn, c-Src and Syk as assessed by phage display. J. Mol. Biol. 1996, 260, 664–677. [CrossRef][PubMed] 46. Lee, T.R.; Niu, J.; Lawrence, D.S. The Extraordinary Active Site Substrate Specificity of pp60c src: A multiple − specificity protein kinase. J. Biol. Chem. 1995, 270, 5375–5380. [CrossRef][PubMed] 47. Glenney, J.R.; Zokas, L. Novel tyrosine kinase substrates from Rous sarcoma virus-transformed cells are present in the membrane skeleton. J. Cell Biol. 1989, 108, 2401–2408. [CrossRef][PubMed] 48. Cabail, M.Z.; Chen, E.I.; Koller, A.; Miller, W.T. Auto-thiophosphorylation activity of Src tyrosine kinase. BMC Biochem. 2016, 17, 13. [CrossRef][PubMed] 49. Lorsch, J.R.; Herschlag, D. The DEAD Box Protein eIF4A. 1. A Minimal Kinetic and Thermodynamic Framework Reveals Coupled Binding of RNA and Nucleotide. Biochemistry 1998, 37, 2180–2193. [CrossRef] [PubMed] 50. Kuai, L.; O’Keeffe, T.; Arico-Muendel, C. Randomness in DNA Encoded Library Selection Data Can Be Modeled for More Reliable Enrichment Calculation. SLAS Discov. Adv. Life Sci. 2018, 23, 405–416. [CrossRef] [PubMed] 51. Zimmermann, G.; Li, Y.; Rieder, U.; Mattarella, M.; Neri, D.; Scheuermann, J. Hit-Validation Methodologies for Ligands Isolated from DNA-Encoded Chemical Libraries. Chembiochem 2017, 18, 853–857. [CrossRef] [PubMed] 52. Barker, S.; Kassel, D.B.; Weigl, D.; Huang, X.; Luther, M.; Knight, W.B. Characterization of pp60c-src Tyrosine Kinase Activities Using a Continuous Assay: Autoactivation of the Enzyme Is an Intermolecular Autophosphorylation Process. Biochemistry 1995, 34, 14843–14851. [CrossRef][PubMed] 53. Masterson, L.R.; Mascioni, A.; Traaseth, N.J.; Taylor, S.S.; Veglia, G. Allosteric cooperativity in protein kinase A. Proc. Natl. Acad. Sci. USA 2008, 105, 506–511. [CrossRef][PubMed] 54. Vogtherr, M.; Saxena, K.; Hoelder, S.; Grimme, S.; Betz, M.; Schieborr, U.; Pescatore, B.; Robin, M.; Delarbre, L.; Langer, T.; et al. NMR Characterization of Kinase p38 Dynamics in Free and Ligand-Bound Forms. Angew. Chemie Int. Ed. 2006, 45, 993–997. [CrossRef][PubMed] 55. Klein, T.; Vajpai, N.; Phillips, J.J.; Davies, G.; Holdgate, G.A.; Phillips, C.; Tucker, J.A.; Norman, R.A.; Scott, A.D.; Higazi, D.R.; et al. Structural and dynamic insights into the energetics of activation loop rearrangement in FGFR1 kinase. Nat. Commun. 2015, 6, 7877. [CrossRef][PubMed] 56. Pucheta-Martínez, E.; Saladino, G.; Morando, M.A.; Martinez-Torrecuadrada, J.; Lelli, M.; Sutto, L.; D’Amelio, N.; Gervasio, F.L. An Allosteric Cross-Talk Between the Activation Loop and the ATP Binding Site Regulates the Activation of Src Kinase. Sci. Rep. 2016, 6, 24235. [CrossRef][PubMed] 57. Tong, M.; Pelton, J.G.; Gill, M.L.; Zhang, W.; Picart, F.; Seeliger, M.A. Survey of solution dynamics in Src kinase reveals allosteric cross talk between the ligand binding and regulatory sites. Nat. Commun. 2017, 8, 2160. [CrossRef][PubMed] 58. Cowan-Jacob, S.W.; Fendrich, G.; Manley, P.W.; Jahnke, W.; Fabbro, D.; Liebetanz, J.; Meyer, T. The Crystal Structure of a c-Src Complex in an Active Conformation Suggests Possible Steps in c-Src Activation. Structure 2005, 13, 861–871. [CrossRef] Molecules 2019, 24, 2764 19 of 19

59. Wendt, M.K.; Smith, J.A.; Schiemann, W.P. Transforming growth factor-beta-induced epithelial-mesenchymal transition facilitates epidermal growth factor-dependent breast cancer progression. Oncogene 2010, 29, 6485–6498. [CrossRef] 60. Balanis, N.; Wendt, M.K.; Schiemann, B.J.; Wang, Z.; Schiemann, W.P.; Carlin, C.R. Epithelial to mesenchymal transition promotes breast cancer progression via a fibronectin-dependent STAT3 signaling pathway. J. Biol. Chem. 2013, 288, 17954–17967. [CrossRef] 61. Armstrong, R.N.; Kondo, H.; Kaiser, E.T. Cyclic AMP-dependent ATPase activity of bovine heart protein kinase. Proc. Natl. Acad. Sci. USA 1979, 76, 722–725. [CrossRef][PubMed] 62. Paudel, H.K.; Carlson, G.M. The ATPase activity of phosphorylase kinase is regulated in parallel with its protein kinase activity. J. Biol. Chem. 1991, 266, 16524–16529. [PubMed] 63. Denton, K.E.; Krusemark, C.J. Crosslinking of DNA-linked ligands to target proteins for enrichment from DNA-encoded libraries. Med. Chem. Commun. 2016, 7, 2020–2027. [CrossRef][PubMed] 64. DeAngelis, M.M.; Wang, D.G.; Hawkins, T.L. Solid-phase reversible immobilization for the isolation of PCR products. Nucleic Acids Res. 1995, 23, 4742–4743. [CrossRef][PubMed] 65. Satz, A.L.; Cai, J.; Chen, Y.; Goodnow, R.; Gruber, F.; Kowalczyk, A.; Petersen, A.; Naderi-Oboodi, G.; Orzechowski, L.; Strebel, Q. DNA Compatible Multistep Synthesis and Applications to DNA Encoded Libraries. Bioconjug. Chem. 2015, 26, 1623–1632. [CrossRef][PubMed] 66. Denton, K.E.; Wang, S.; Gignac, M.C.; Milosevich, N.; Hof, F.; Dykhuizen, E.C.; Krusemark, C.J. Robustness of In Vitro Selection Assays of DNA-Encoded Peptidomimetic Ligands to CBX7 and CBX8. SLAS Discov. 2018, 23.[CrossRef] 67. Kim, D.; Jetson, R.R.; Krusemark, C.J. A DNA-assisted immunoassay for enzyme activity via a DNA-linked, activity-based probe. Chem. Commun. 2017, 53, 9474–9477. [CrossRef][PubMed] 68. Seeliger, M.A.; Young, M.; Henderson, M.N.; Pellicena, P.; King, D.S.; Falick, A.M.; Kuriyan, J. High yield bacterial expression of active c-Abl and c-Src tyrosine kinases. Protein Sci. 2005, 14, 3135–3139. [CrossRef]

Sample Availability: Samples of all off-DNA synthesized compounds are available from the authors.

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).