This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes.
Article Cite This: ACS Omega 2019, 4, 775−784
http://pubs.acs.org/journal/acsodf
Conceptional Design of Self-Assembling Bisubstrate-like Inhibitors of Protein Kinase A Resulting in a Boronic Acid Glutamate Linkage Janis Müller,†,§,∥ Romina A. Kirschner,‡,§,⊥ Armin Geyer,*,‡ and Gerhard Klebe*,† †
Institute of Pharmaceutical Chemistry, Philipps-University Marburg, Marbacher Weg 6, 35032 Marburg, Germany Department of Chemistry, Philipps-University Marburg, Hans-Meerwein-Straße 4, 35032 Marburg, Germany
‡
Downloaded via 109.94.221.209 on January 19, 2019 at 06:47:53 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
S Supporting Information *
ABSTRACT: The spontaneous esterification of boronic acids with polyols provides a promising opportunity to generate selfassembled bisubstrate-like inhibitors within the binding pocket of cAMP-dependent protein kinase (PKA). As a first substrate component, we designed amino acids, which have either a boronic acid or ribopyranose side chain and introduced them to the substrate-like peptide protein kinase inhibitor (PKI). The second component was derived from the active-site inhibitor Fasudil, which was functionalized with phenylboronic acid. NMR spectroscopy in dimethylsulfoxide proved spontaneous reversible condensation of both components. Reinforced by the protein environment, both separately bound substrates were expected to react via boronic-ester formation bridging the two binding sites of PKA. Multiple crystal structures of PKA with bound PKIs, positionally modified with residues such as a ribopyranosylated serine and threonine or a phenylboronic acid attached to lysine via amide bonds, were determined with the phenylboronic acid-linked Fasudil. Although PKA accepts both inhibitors simultaneously, the expected covalent attachment between both components was not observed. Instead, spontaneous reaction of the terminal boronic acid group of the modified Fasudil with the carboxylate of Glu127 was detected once the latter residue is set free from a strong salt bridge formed with arginine by the original peptide inhibitor PKI. Thus, the desired self-assembly reaction occurs spontaneously in the protein environment by an unexpected carboxylic acid boronate complex. To succeed with our planned self-assembly reaction between both substrate components, we have to redesign the required reaction partners more carefully to finally yield the desired bisubstrate-like inhibitors in the protein environment.
■
INTRODUCTION Dysregulation of protein kinase function can lead to the development of disease situations because of the important role of these enzymes in cell-cycle regulation and signal transduction. As a consequence, cancer may be induced in eukaryotic cells.1 For this reason and because 2% of the human genome consists of protein kinases, these enzymes have emerged as one of the major therapeutic targets to be functionally modulated by kinase-specific inhibitors.2 Protein kinase A (PKA) is regarded as the best understood enzyme of this protein superfamily and widely used as a representative prototype.3 The development of bisubstrate-like inhibitors falls into the field of supramolecular chemistry and is expected to boost binding affinity with respect to the separate individual components for entropic reasons as first suggested by Jencks and subsequently demonstrated by several case studies.4−10 Both molecular portions to be connected by a linker will simultaneously mimic the natural substrates or substrate-like © 2019 American Chemical Society
inhibitors by reducing the number of translational and rotational degrees of freedom, at the same time keeping or even enhancing the number of interactions to be experienced at both binding sites.11 Furthermore, most likely, selectivity is enhanced for a given enzymatic target by such bisubstrate-like inhibitors compared to the smaller and chemically simpler single-site inhibitors.11−13 PKA was chosen as a test system for the synthesis and development of such bisubstrate-like inhibitors. In a previous study, this system has already been used for the development of bisubstrate analogous inhibitors. Pflug et al.14 and Lavogina et al.15 developed adenosine-analogous oligo-arginine conjugates and studied their binding properties by crystal structure analysis. Different to our strategy, the ligands reported in these studies14,15 had a covalent linkage between both substrate-like Received: September 12, 2018 Accepted: December 7, 2018 Published: January 9, 2019 775
DOI: 10.1021/acsomega.8b02364 ACS Omega 2019, 4, 775−784
ACS Omega
Article
Figure 1. Overview of the conceptual design of this study. We planned the formation of a reversible covalent linkage of the boronic acid-modified inhibitor Fasudil (upper part (A)) with the pyranose-containing peptidomimetic substrate (ribopyranosylated serine or threonine residue) in PKI. Alternatively, the Lys-side chain was functionalized with a boronic acid [lower part (B)] and expected to react via spontaneous esterification with the ribofuranose of ATP. Both strategies were supposed to lead to the shown covalently tethered supramolecular structures possibly driven by the entropically favored release of water molecules. The displayed structures were constructed and energy-minimized17 to design the modified peptides for the mutagenesis experiments.
parts already introduced by chemical synthesis prior to protein binding. In our concept, we intended to produce the bisubstrate-like inhibitors based on the spontaneous formation of a reversible covalent linkage instead of other approaches using irreversible reactions such as click chemistry.16 The reversible linkage was planned between the natural substrate adenosine phosphate or an inhibitory scaffold designed for this site and a substrate-like peptide stretch mimicking the protein sequence to be phosphorylated [protein kinase inhibitor (PKI), in the following residues of PKA is usually numbered; PKI residues are numbered as superscripts]. The mutual linkage between both portions was intended to be formed upon esterification of a boronic acid portion. Two design strategies have been followed in the current contribution. First, a boronic acid-modified ligand derived from the known PKA inhibitor Fasudil was planned to react with a glycosylated amino acid incorporated into the substratemimicking PKI sequence (Figure 1, upper part). In a second approach, we incorporated an amino acid decorated by a terminal boronic acid group into PKI. The latter group was then intended to react with the 2′ and 3′ OH groups of the adjacent ribose moiety of the ATP substrate (Figure 1, lower part). Our intention was that the linkage of both inhibitory portions will result by boronic-ester formation reinforced by the spatial proximity and the template forcing effect of the surrounding PKA. Possibly, the entropic advantage resulting from the release of water during the reaction of the diol and the boronic acid component will overcome the disfavored esterification in aqueous solution. Under the latter conditions, the overwhelming presence of water molecules competes with this reaction step. Our hypothesis was supported by the observation that the desired esterification is spontaneously
observed in dimethylsulfoxide (DMSO) solution by NMR spectroscopy. The idea of using glycosylated amino acids and boronic acids as building blocks for the linkage between a substrate peptide and an inhibitor or ATP was driven by the observation that certain sugars have a strong affinity toward boronic acids with respect to esterification reactions.18,19 This tendency for ester formation of boronic acids with 1,2- and 1,3-diols was already described by Magnanini et al. in 1890 and later expanded by Böeseken et al. in 1913.20,21 They were able to determine saccharide configurations by measuring the change in conductivity and acidity by titration of boric acid to the sugar-containing solution. Boronic esters have found applications in the dynamic covalent assembly of supramolecular structures.22−24
■
RESULTS AND DISCUSSION Synthesis. Lewis acid-mediated glycosylation reactions with tetra-O-acetyl-D-ribopyranose (1, Scheme 1) transformed the hydroxyl amino acids N-(9H-fluoren-9-ylmethoxy)carbonyl-L-serine (Fmoc-Ser) and Fmoc-threonine (FmocThr), respectively, into the ribopyranosylated amino acid building blocks RbS (2) and RbT (3, the capital letter indicates the natural L-configuration, similar to the three-letter code established for amino acids).25 The modified amino acids were used in manual or automated synthesis for the solid-phase assembly of PKI derivatives as 20mer peptides 4−10. All analytical data are given in the Supporting Information. We chose ribose in its pyranoid conformation (1) because of the high affinity of the axial−equatorial−axial trihydroxy arrangement, which is able to bind boronic acids (Scheme 1A).26 Sidechain acylation of Lys ε-NH2 with 3-carboxyphenylboronic acid yielded PKI containing the amino acid BoK with a side776
DOI: 10.1021/acsomega.8b02364 ACS Omega 2019, 4, 775−784
ACS Omega
Article
Scheme 1. (A) Ribopyranosylation of Fmoc-Ser and Fmoc-Thr with Trimethylsilyl Triflate and Tin Tetrachloride, Respectively, Yielded the Amino Acids 2 and 3, which Were Then Used in Solid-Phase Peptide Synthesis of PKI Derivatives 4−10; (B) 1-(4,4-Dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde) Protecting Group was Utilized in the Synthesis of Another PKI Variant 11. Treatment of the Resin-Bound Peptide with Hydrazine Liberated the Lys ε-NH2 Which was Subsequently Acylated with meta-Phenylboronic Acid in the Presence of N,N,N′,N′-Tetramethyl-O-(1H-benzotriazol-1yl)uronium Hexafluorophosphate, 1-Hydroxybenzotriazole, and the Base Diisopropylethylamine (DIPEA) in Dimethylformamide (DMF); the Final Side-Chain Deprotection and Cleavage of the Peptide from the Resin Was Performed Under Acidic Conditions
chain phenylboronic acid (11, Scheme 1B). RbS, RbT, and BoK mutations were introduced at different positions near the active site of PKI. Further details are described in the Methods section. As shown by Glass et al., the minimal length of the PKI inhibitory peptide to achieve binding affinity in the nanomolar range has to comprise the residues 6−22 apart from the serine/ alanine replacement at the catalytic site.27 Amino acids Tyr7, Phe10, Asn20, and Ile22 of PKI were described as essential for the interaction with the protein, as well as the arginine residues Arg15/18/19.28,29 We prepared all peptides which contained systematically the ribosylated amino acids between position 13 (4) and the phosphorylation site 21 (10). The positions used for the replacement by RbS and RbT were selected by measuring the distance between PKI and the ATP-binding site in the crystal structures of PKA which contained PKI. It was expected that the modified PKI sequences of Scheme 1 alone have low affinities for PKA without an additional active-site inhibitor. They suggested favorable distances between the sugar moiety and the boronic acid attached to an inhibitor scaffold for the desired esterification reaction. Fasudil (Scheme 2) served as the chemical lead because of its potency for PKA, which falls into the nanomolar range.30−32
Scheme 2. Inhibitor 12 Derived from the Known Inhibitor Fasudil and Phenylboronic Acid
Reductive amination of Fasudil with formylphenylboronic acid yielded boronic acid 12. NMR Spectroscopy. The different PKI variants were characterized by NMR spectroscopy to assess whether they adopt random coil structures or a preferred conformation. As indicated by the resolved crystal structures, PKI adopts αhelical geometry next to the N-terminus of the peptide in the protein-bound state.33 Nevertheless, this conformation could 777
DOI: 10.1021/acsomega.8b02364 ACS Omega 2019, 4, 775−784
ACS Omega
Article
Figure 2. Exemplary and complete assignment of the protons of peptide 7 using spectra [600 MHz, 300 K, potassium phosphate buffer (50 mM, pH 3.0)/D2O 9:1]. Highlighted are the NH− and the α-proton areas of the spectra.
microscale thermophoresis (MST) (Methods section).34 In parallel, we tried to crystallize the different peptide variants and inhibitor 12 or ATP with PKA to elucidate the adopted binding modes (Methods section). To develop appropriate conditions to perform the MST assay and to validate the obtained results, we first determined the binding constants of wild-type PKI and Fasudil. The determined affinity data fall roughly into the same range as obtained by Cheng et al.31 and Wienen-Schmidt et al.32 in the literature; however, both groups applied alternative assays. To our experience, such systematic deviations which can shift the binding data on absolute scale by 1 or 2 orders of magnitude are easily observed between assay following different measurement principles; however, the relative differences remain similar. For the different PKI variants containing either RbS or RbT at the different sequential positions, the following binding data could be recorded using the MST assay (Table 1).
not be detected for the isolated peptide in solution by NMR. Figure 2 shows an example of the obtained 1H NMR spectra of peptide 7. The spontaneous esterification between ribosylated peptides and boronic acids was tested in an apolar solvent before the affinity was assessed against PKA. PKI derivative 7 PKI(RbS18)5−22 was investigated for its ability to bind the inhibitor 12 in deuterated DMSO. The slow chemical exchange provided individual signal sets in the 1H NMR because of the slow chemical exchange between the boronic ester, peptide, and boronic acid. The ester was identified by (i) the downfield shift of the 3-OH proton of ribopyranose in DMSO, (ii) the characteristic chemical shift of its anomeric CH group around 100 ppm in the heteronuclear single quantum coherence (HSQC) spectra, and (iii) its decreased anomeric 3 J coupling constant. The signals of the unbound sugar disappeared and thus indicated complete conversion. An expansion of the anomeric region from the HSQC spectrum of peptide 7 and inhibitor 12 is shown in Figure 3.
Table 1. Binding Data Obtained in the MST Assay PKI variant 5−24
PKI PKI5−22 PKI(RbS13)5−22 (4) PKI(RbT16)5−22 (5) PKI(RbS17)5−22 (6) PKI(RbS18)5−22 (7) PKI(RbS19)5−22 (8) PKI(RbS20)5−22 (9) PKI(RbS21)5−22 (10) PKI(BoK18)5−22 (11) 12
Figure 3. Expansion from the HSQC spectrum (600 MHz, 300 K, DMSO-d6) of the titration of peptide PKI(RbS18)5−22 (7) with the inhibitor 12. The relative intensity of the signals shows the amount of boronic ester.
KD from MST (μmol·L−1) 0.144 ± 0.03 4.32 ± 0.89 2.67 ± 0.35 26.00 ± 4.65 93.98 ± 82.59 >100 >100 29.60 ± 10.00 >100 >100 5.86 ± 2.75
We observed low but detectable binding affinities for the shortened PKI variants. The position of the variation was crucial for the change in binding affinity. Most variations were tolerated without substantial loss in affinity; however, once the variations occurred close to position 18, the affinity decreased significantly to a threshold where binding could no longer be measured by MST. This was in accordance with findings from Scott et al.35 where the presence of Arg18 was reported to be essential for the binding affinity. The borylated amino acid BoK was incorporated at position 18 into the sequence of PKI5−22 (11). Unfortunately, in the
Affinity Determination by Microscale Thermophoresis. After characterizing the self-assembly process in the organic solvent, we investigated whether also in the protein environment a similar esterification across the substratebinding sites could be observed. The binding experiments to determine affinities of the bisubstrate−inhibitor system and PKA were performed in aqueous buffer solution using 778
DOI: 10.1021/acsomega.8b02364 ACS Omega 2019, 4, 775−784
ACS Omega
Article
Table 2. Protein Data Bank (PDB) Codes of Obtained Crystal Structures peptide PKI5−22 PKI(RbS13)5−22 (4) PKI(RbT16)5−22(5) PKI(RbS17)5−22(6) PKI(RbS18)5−22(7) PKI(BoK18)5−22(11) PKI(RbS19)5−22(8) PKI(RbS20)5−22(9) PKI(RbS21)5−22(10)
soloa
Fasudil
6EH0 6ERV 5OUA n.d.b
6EM2 6ERW
5NTJ 5OUS 6ERU
6I2A 6I2C 5NW8
5O0E
12
AMP
ADP
ATP
5OL3 6EGW 6I2B 6I2D n.d.b 6ERT
6EH2 n.d.b
6EM6
n.d.b
6ESA
6EM7
6EMA
5O5M 5OT3
a Solo indicates binary complexes of PKA and modified PKI. All other complexes are ternary complexes of PKA with modified PKI and a ligand in the ATP site (Fasudil, 12, AMP, ADP, and ATP). bStructures marked by n.d. were obtained but not deposited because of pronounced disorder in important areas next to the substrate recognition sites.
Figure 4. Comparison of complexes between PKA and PKI5−24 (left, PKA in blue, wild-type PKI5−22 in green, PDB-code: 2GFC) and PKAPKI(RbS13)5−22 (4) (right, PKA orange, PKI yellow, PDB-code: 5OUA). The superposition of both structures is shown in the center. The PKI variants are highlighted by a red ellipsoid.
A comparison between the binding modes of all PKI variants studied in this contribution along with the geometry of PKI5−24 reported in the literature is shown in Figure 5.
MST assay, no binding affinity could be recorded for the interaction of this peptide with PKA, neither in the absence nor in the presence of ATP. These results match with our findings described above and data in the literature,14,15,27,35 indicating that the replacement of Arg18 in the wild-type PKI results in a dramatic loss of affinity. Crystal Structure Analysis of the Peptide−Boronic Acid Derivatives. To relate the above-described affinity data with the adopted binding modes of the mutated PKIs and the added ligand 12 and ATP and to elucidate whether any spontaneous reaction of the boronic acid moiety had occurred, we tried to crystallize the various complexes (Methods section). For those where we obtained suitable crystals, the corresponding crystal structures were determined, partly with and without a bound ligand in the ATP-binding site (Table 2 and Supporting Information). Crystallization for the various complexes was performed according to a general cocrystallization protocol, even though in detail, the concentrations and particularly the crystallization time had to be adjusted for each experiment. The individual conditions and details on the data collections and refinements are given in the Supporting Information (Sections 3.4 and 3.5). Binding Modes of the PKI Variants. Overall, the multiple crystal structures indicate only minor modifications of the bound PKI variants. Individual shifts, local disorder, or slight unwinding of the helical part of PKI can be related to an increasing steric demand of the incorporated, chemically modified amino acids at the various positions of the PKI sequence. An example for such a shift is shown in Figure 4.
Figure 5. Binding modes of all studied PKI variants, depicted as cartoon drawings. In all examples, the surrounding PKA has been omitted for clarity. The binding mode of PKI5−24 (orange) has been extracted from the corresponding PDB entries according to following color codes applied to the various structures: 6ERV brown, 5OUA magenta, 5NTJ green, 6ERU dark green, 6I2C yellow, 5OT3 gray, and 5OUS red. The peptide stretch found in the complex structure of PKI(RbT16)5−22 (5), not deposited in the PDB according to its only partly defined electron density, is shown in blue.
Binding Modes of the Boronic Acid-Modified Fasudil 12. With respect to the adopted orientation of 12 in the various complex structures, we observed, apart from those showing a modified amino acid in position 18, a rather conserved binding mode. In these complexes, the fasudil moiety is virtually superimposable. Only the conformation of the seven-membered ring and the attached phenylboronic acid 779
DOI: 10.1021/acsomega.8b02364 ACS Omega 2019, 4, 775−784
ACS Omega
Article
Figure 6. Binding modes of inhibitor 12 in the various studied complexes; for clarity, only 12 is shown together with the observed 2mFo − Fcelectron densities contoured by the blue mesh at 1σ. The atoms of PKI and PKA were omitted in the images. All phenylboronic acids are oriented toward Asp184 and involve this residue in a charge-assisted hydrogen bond, sometimes as a dual and sometimes as a single contact. (A) PKAPKI5−22, PDB-code 5OL3; (B) PKA-PKI(RbS13)5−22 (4) PDB-code 6EGW; (C) PKA-PKI(RbS20)5−22 (9) PDB-code 5O5M; (D) PKAPKI(RbT16)5−22 (5) PDB-code 6I2B; (E) PKA-PKI(RbS17)5−22 (6) PDB-code 6I2D; and (F): PKA-PKI(RbS21)5−22 (10) PDB-code 5OT3.
adopt slightly different orientations to establish a chargeassisted hydrogen bond to the side-chain carboxylate group of Asp184. As the observed interaction geometries of these Hbonds show, the steric requirements of the incorporated, modified amino acids in the adjacent PKI variants take a slight influence on the quality and strength of this contact. Details are displayed in Figure 6. The hydrogen bond are listed in Table 3. Table 3. Hydrogen Bond Length of 12 to Asp184a complex 5−22
PKA-PKI -12 PKA-PKI(RbS13)5−22(4) + 12 PKA-PKI(RbT16)5−22(5) + 12 PKA-PKI(RbS17)5−22(6) + 12 PKA-PKI(RbS20)5−22(9) + 12 PKA-PKI(RbS21)5−22(10) + 12
bond-length 1 (Å)
bond-length 2 (Å)
3.3 3.3 3.2 3.0 3.3 3.3
3.2 3.5 3.5 3.3 3.6
a
Distances between the OH groups at boron and the closest carboxylate oxygen of Asp184.
Figure 7. Electron density (2mFo − Fc and mFo − Fc densities at 1σ, blue or green mesh, resp.) found in the complex of PKA with PKI(RbS18)5−22 (7) and inhibitor 12 in the area of RbS18, Glu127, and the phenylboronic acid terminus. The density suggests two orientations of the phenylboronic acid, either toward Asp184 and in opposite direction toward RbS18 and the carboxylate group of Glu127. The accuracy of the observed density does not allow for assigning a covalent attachment between the boronic acid group and Glu127 (Figure 8). Most likely, hydrogen bonds are experienced by this group with the adjacent ribo-sugar OH groups and the side-chain carboxylate of Glu127.
Complexes with PKI Variants Modified in Position 18. As expected, most promising for our strategy to link the inhibitory peptide PKI with the ligand accommodating the ATP site is the position 18 in the PKI sequence. Even though the measured binding affinity of some peptide variants demonstrate strongly reduced affinity, we obtained crystal structures with these variants. Unfortunately, the complex of PKI(RbS18)5−22 (7) with inhibitor 12 exhibits pronounced disorder, which makes interpretation of the electron density difficult (Figure 7). With some care, 12 can be placed in two competitive orientations into the density. The first corresponds to the usual orientation showing the phenylboronic acid groups adjacent to Asp184 and it interacts via hydrogen bonds with this residue. Nevertheless, the density also indicates a second conformer placing the boronic acid group in opposing direction next to the carboxylate group of Glu127 and adjacent to the ribose ring of PKI(RbS18)5−22 (7). Undoubtedly, the sugar moiety of the latter residue did not undergo the desired covalent attachment with the boronic acid group. This is evident by the pronounced disorder and the distances found for the model assigned to the density. Most likely, only weak hydrogen bonds are formed. Whether any further contacts are established to Glu127 is difficult to assess (s. below). The latter Glu residue is usually involved in a salt-bridge with Arg18 in the sequence of natural substrate-like inhibitor PKI. In the present PKI(RbS18)5−22 (7) complex, the Arg18 residue is replaced and the liberated carboxylate group of Glu127 is ready for other
contacts. The loss of the Arg18−Glu127 salt-bridge also explains the dramatic drop in affinity once this crucial residue is replaced by another amino acid in PKI. However, only if this strong salt-bridge is sacrificed, for example, as a consequence of the Arg18-by-RbS18 replacement, will the carboxylate group of Glu127 become available for alternative contacts. Spontaneous Bond Formation of Boronic Acid 12 with Glu127. The latter assumption is strongly supported by the crystal structure of PKI(BoK18)5−22 (11) with 12. As a matter of fact, the atoms of the attached BoK18 residue are only defined in the electron density until Cβ. Obviously, the attached terminal functional group of this residue avoids short contacts with PKA or 12. Instead, 12 adopts the abovesuggested second conformation toward the front and its boronic acid group is exclusively placed with full occupancy next to the carboxy group of the liberated Glu127. Remarkably, the carboxylate now performs a nucleophilic attack onto boron transforming the latter atom into tetrahedral geometry (Figure 8). The covalent attachment spontaneously formed between 780
DOI: 10.1021/acsomega.8b02364 ACS Omega 2019, 4, 775−784
ACS Omega
Article
geometry at the ATP-binding site appropriate to undergo the desired covalent attachment with the adjacent ribo-sugar moiety of the peptide component. One option to test for a more competent binding geometry would be to open the central seven-membered ring of Fasudil by replacing it with a methylene linker of varying lengths to launch the terminal boronic acid group appropriately for the planned reaction step. The enhanced flexibility of such a linker chain will provide the advantage of a better adaptability to the requirement of the binding pocket. Nevertheless, as a significant loss in preorganization of the ligand to be bound will reduce the binding potency, it might be necessary to seek for alternative scaffolds that allow both correct placement of the boronic acid group along with sufficient preorganization of the ATP-site ligand. Our future design will follow such concepts to develop an improved ATP-like substrate component with promising geometry for the planned self-assembly reaction.
the ligand and the protein is a carboxylic acid-boronate contact, which is a novelty in inhibitor design.
■
Figure 8. In the complex of PKI(BoK18)5−22 with 12, the carboxylate group of Glu127 is no longer involved into a contact to PKI. Thus, the terminal boronic acid group of the latter inhibitor attaches covalently to the carboxylate group of Glu127 and leads to a boronate complex. PKA is shown as a green cartoon, and atoms of the PKI peptide (lower right) are shown in yellow.
METHODS Protein Production and Purification. The catalytic subunit alpha of PKA from Chinese hamster (Cricetulus griseus; UniProt ID P25321, 98% sequence identity with the human isoform) with an added His6 tag was recombinantly produced in Escherichia coli BL21(DE3)pLysS cells from Life technologies using a plasmid from Kudlinzki et al.36 The cells were grown in ZYM5025 medium containing the antibiotics ampicillin (100 mg/L) and chloramphenicol (35 mg/L). The cell culture was grown at 37 °C overnight, afterward induced with 1 mM IPTG, and grown at 20 °C for 60 h. Subsequently, the cells were harvested by centrifugation, flash-frozen with liquid nitrogen, and stored at −80 °C overnight. The cell pellets were resuspended with a 50 mM NaH2PO4/Na2HPO4 buffer (pH 8) containing 500 mM sodium chloride, 10 mM imidazole, and small amounts of DNAse II and protease inhibitor cocktail tablets from Roche. The cells were disrupted with Emulsiflex C3 from Avestin and centrifuged. The PKA Cunit was purified from the supernatant via immobilized metalion affinity chromatography (HisTrap HP from GE Healthcare) and eluted with a gradient with 50 mM NaH2PO4/ Na2HPO4 buffer (pH 8) containing 500 mM sodium chloride and 500 mM imidazole. The His6 tag was removed by TEV protease digestion while dialyzing the protein against a 50 mM NaH2PO4/Na2HPO4 buffer (pH 8) containing 100 mM sodium chloride and 5 mM dithiothreitol (DTT) at 4 °C for 72 h. The PKA was purified via immobilized metal-ion affinity chromatography (HisTrap HP from GE Healthcare). The flow-through was dialyzed against a 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (pH 7) containing 7 mM sodium chloride and 5 mM DTT at 4 °C overnight. The different phosphorylation states of the recombinantly produced PKA C-unit were separated via cation-exchange chromatography (Mono S, GE Healthcare) and a sodium chloride gradient. The different phosphorylation states were collected separately, flash-frozen with liquid nitrogen, and stored at −80 °C until further use. Peptide Synthesis. ortho-Chlorotritylchloride resin (1.60 mmol/g) was mixed with the Fmoc-protected amino acid (1.50 equiv) and DIPEA (6.00 equiv) in DMF (10 mL/g resin) and stirred for 5 h. After washing the resin with DMF, methanol, and dichloromethane three times, the resin was treated with a mixture of dichloromethane/methanol/DIPEA (80:15:5) two times for 30 min and washed again three times with DMF, methanol, and dichloromethane before it was dried
Thus, even though the PKI(BoK18)5−22 (11) + 12 complex does not show a self-assembly between PKI and the occupant of the ATP site, the complex produces instead a covalent attachment between the boronic acid group and PKA residue Glu127. Interestingly, in contrast to the binding mode in the PKI(RbS18)5−22 (7) + 12 complex (Figure 8), only one fully populated orientation of the phenylboronic acid side chain is observed and the coordination at boron is definitely tetrahedral. This result suggests that the selected boronic acid group is competent to undergo the expected self-assembly reaction in the protein environment using a nucleophilic attack.
■
CONCLUSIONS In the present study, a novel concept for the development of potent and selective kinase inhibitors has been suggested that aims at the self-assembly of bisubstrate analogue building blocks to form a larger and more potent inhibitor via covalent attachment in the protein environment. Even though we could show that the planned self-assembly step is principally possible, our initial concept suffered from the following aspects. First, the residue in the peptide substrate component, which should carry the group planned for the self-assembly reaction with the ATP site substrate component, has to be placed in position 18 of the peptide sequence. In the substrate-analogue peptide inhibitor PKI, an arginine is found at that position, which forms a crucial salt-bridge to Glu127 of PKA. Any displacement of this residue results in a dramatic loss of affinity. This finding matches well with the results of Pflug et al.14 and Lavogina et al.15 In their development of potent adenosine-analogous oligo-arginine conjugates as bisubstrate mimetics, the introduction of more than two arginine residues at the terminal end along with extended linkers revealed improved affinity toward PKA. As the crystal structures (PDB entries: 3AG9, 3AGL, 3AGM) show, one of the crucial arginine residues interacts with Glu127. Possibly, in our concept, the loss of this interaction to Glu127 can be compensated by the favorite self-assembly reaction step. Second, our designed inhibitor 12 does not yet adopt a 781
DOI: 10.1021/acsomega.8b02364 ACS Omega 2019, 4, 775−784
ACS Omega
Article
of 10 mM Mega 8 solution, and 6 μL of 10 mM peptidic ligand solution. For crystallization with hinge binders, the latter was added as a solid material so that the final concentration of the hinge binder in the solution was 5 mM. This master-mix was centrifugated for 15 min. Crystals were grown by the vapordiffusion method at 4 °C in 3 μL sitting drops of the mastermix against 400 μL of methanol/water solutions with methanol concentrations of 14−23% (volume fraction). Methanol concentrations and crystallization time were individually adjusted (Supporting Information, Table S1). Crystals without peptidic ligands were produced as described by Siefker.39 The crystals were harvested via a cryo loop and transferred into 70% (volume fraction) of 100 mM Mes−Bis−Tris buffer (pH 6.9) containing 1 mM DTT, 0.1 mM sodium EDTA, and LiCl and methanol concentrations analogous to the conditions in the crystal drop-mixed with 30% (volume fraction) 2methyl-2,4-pentanediol (MPD, cryo buffer). After a few seconds, the crystal was harvested with a cryo loop again and flash-frozen in liquid nitrogen. Crystal Soaking. Crystals without peptidic ligands were prepared as described by Siefker39 and harvested analogously to the other crystals. However, in these cases, the abovementioned cryo buffer contained 10 mM of the hinge binder and the crystals were harvested from cryo buffer after 2 hours. Afterward the crystals were flash-frozen in liquid nitrogen. Data Collection, Data Reduction, Phasing, and Refinement. Data collection was performed at ESRF in Grenoble (Beamline ID29),40 EMBL in Hamburg (Beamline P13),41 und BESSY II (Beamline 14.1, 14.2 und 14.3) in Berlin.42 The required number of images, degree of rotation per image, and exposition time was calculated by taking test images before and after a 90° rotation and following the proposed strategy as computed by Mosflm.43 Data indexing, scaling, and integration was performed with XDS.44 Afterward, a molecular replacement using an in-house structure of the PKA C-unit was performed using PHASER.45 The structures were refined by iterative cycles with Phenix,46 while model building was performed with Coot.47 Crystallographic Tables summarizing the measuring conditions, unit cell dimensions, data collection, and refinement statistics of crystal structure analyses can be found in the Supporting Information.
under vacuum. The loading of the used resins was estimated to be between 0.50 and 0.60 mmol/g by UV spectroscopy at 289 and 300 nm after cleaving the Fmoc protecting group with 20% piperidine in DMF for 20 min. Peptides were synthesized by the Fmoc strategy on a microwave-assisted peptide synthesizer (Liberty Blue, CEM). The resin with a scheduled quantity of 0.1 mmol (1.00 equiv) loaded Fmoc-amino acid ran through the following cycles: Fmoc-deprotection: T = 50 °C, Pmicrowave = 30 W, t = 210 s with piperidine (20 wt % in DMF, 3.00 mL/deprotection). Amino acid coupling for amino acids other than Nα-Fmoc-Nω-(2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl)-L-arginine Fmoc-Arg(Pbf)-OH: T = 50 °C, Pmicrowave = 30 W, t = 600 s, 0.2 m Fmoc-protected amino acid in DMF, 5.00 equiv, 2.5 mL/coupling, DIC (0.5 m in DMF, 5.00 equiv, 1 mL/coupling), and Oxyma (1 m in DMF, 5.00 equiv, 0.5 mL). For Fmoc-Arg(Pbf)-OH: T = 25 °C, Pmicrowave = 0 W, t = 1500 s, than T = 50 °C, Pmicrowave = 35 W, t = 660 s. Resin cleavage: resin cleavage was performed with a mixture of trifluoroacetic acid, phenol, H2O, and triisopropylsilane in a ratio of 88:4:4:4 for 3 h. Peptides were precipitated from cold diethyl ether (40 mL), washed two to three times with diethyl ether, and lyophilized from water. Acetyl groups of glycosylated amino acids were deprotected on solid phase by washing twice for 30 min with 2% NaOMe in MeOH under argon, and the resin was washed with water, Et2O, three times with DMF, and six times with DCM according to Cai et al.37 The analytical characterization of the individual peptide inhibitors is summarized in the Supporting Information. Measurement of the MST Assay. Solutions of fourfold phosphorylated PKA C-unit (concentration 18 μM) were labeled with a fluorescent group by using a Monolith NT Protein Labeling Kit RED-NHS from Nanotemper technologies.38 After labeling, the protein was resolved in a 50 mM HEPES buffer (pH 7) containing 100 mM NaCl, 10 mM MgCl2, 1 mM TCEP, and 0.05% Tween. The protein solution was diluted to
