Deactivation of a dimeric DNA-binding peptide through a palladium-mediated self-immolative cleavage

Deactivation of a dimeric DNA-binding peptide through a palladium-mediated self-immolative cleavage† Jessica Rodr´ ıguez, ‡Cibr´ an P´ erez-Gonz´ alez, ‡Miguel Mart´ ınez-Calvo, Jes´ us Mosquera and Jos´ e L. Mascare˜ nas * Herein, we describe an approach for the on-demand disassembly of dimeric peptides using a palladiummediated cleavage of a designed self-immolative linker. The utility of the strategy is demonstrated for the case of dimeric basic regions of bZIP transcription factors. While the dimer binds designed DNA sequences with good affinities, the peptide–DNA complex can be readily dismounted by addition of palladium reagents that trigger the cleavage of the spacer, and the release of unfunctional monomeric peptides. In recent years, there has been a lot of progress in the development of uncaging reactions that release active drugs or biopolymers from inactive precursors. 1 The strategy lies on a temporary blocking of a key functional group of an active compound with a caging/protecting group, protection that cancels its biological function. The activity can be eventually recovered by removing the caging pendant, a process that is usually achieved upon application of a light pulse, 2 a chemical trigger 3 or a metal-based reagent. 4 This latter strategy has gained relevance in recent years, owing to its intrinsic catalytic potential and the variety of mechanisms offered by organometallic chemistry. Many of the biological applications of transition metal-mediated bioorthogonal uncaging reactions have relied on the use of allyl or propargyl derivatives as protecting groups, and ruthenium or palladium catalysts (Fig. 1a). 5 In particular, palladium-mediated depropargylation reactions have been extensively used for “gain of function”processes based on the release of bioactive products containing amine or alcohol functionalities. 6 Surprisingly, metal-promoted processes that lead to “loss of function”properties, which could nd important applications in biosupramolecular chemistry, cell biology and/or biomedicine, are essentially unknown. 7 Considering our previous work on the deactivation of dimeric DNA-binding peptides using light, 8 we wondered whether it would be possible to cleave this type of dimers using a metal-mediated protocol. This would be relevant not only in terms of developing a new methodology to regulate DNA binding processes, but also because of proposing a new type of Fig. 1 (a) Representation of the metal-mediated uncaging strategies in chemical biology. (b) Mechanistic proposal for a palladium-mediated dedimerizing cleavage. (c) Illustration of the self-immolative GCN4 dimer, and palladium-mediated uncaging with suppression of the DNA interaction. Centro Singular de Investigaci´ on en Qu´ ımica Biol´ oxica e Materiais Moleculares (CIQUS), Departamento de Qu´ ımica Org´ anica, Universidade de Santiago de Compostela, R´ ua Jenaro de la Fuente s/n, Santiago de Compostela, 15782, Spain. E-mail: joseluis.mas[email protected] †Electronic supplementary information (ESI) available. See DOI: 10.1039/d1ra09180h ‡These authors contributed equally. Cite this: RSC Adv., 2022, 12,3500 Received 19th December 2021 Accepted 11th January 2022 DOI: 10.1039/d1ra09180h rsc.li/rsc-advances 3500 |RSC Adv., 2022, 12, 3500–3504 © 2022 The Author(s). Published by the Royal Society of Chemistry RSC Advances PAPER Open Access Article. Published on 26 January 2022. Downloaded on 1/27/2022 9:30:43 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online View Journal | View Issue uncaging approach based on the cleavage of a linker rather than on the release of a caging group. Although there are some precedents on the uncaging of temporary linked systems, they have been essentially centered on the dual release of two active molecules, rather than on the deactivation of biorelevant processes. 9 Using as reference a report describing the viability of cleaving benzylic alcohols in ortho to arylether derivatives, 10 we reasoned that introducing a propargylic protecting group in this type of phenols might provide for a Pd-mediated release of the pendants (Fig. 1b). Herein we demonstrate the viability of this approach, by describing the synthesis of DNA-binding bZIP-based peptides dimerized through a phenol-like linker that undergoes a selfimmolative cleavage aer treatment with a palladium reagent. This process results in the release of monomeric peptides and suppression of the DNA binding (Fig. 1c). Basic leucine zippers (bZIP) are among the most important families of transcription factors (TFs). They are formed by an N- terminal basic region (br) that makes specic interactions with the major groove of the DNA, and a C-terminal leucine zipper domain that mediates dimerization. 11 Monomeric basic regions are not able to bind their consensus DNA sites with enough affinity, unless they are connected to other DNA binders, 12 or arranged into pre-folded constructs. 13 Alternatively, the DNA binding can be rescued when the basic region peptides are engineered as dimeric systems using suitable synthetic connectors. 14 To implement our on–offDNA binding system, we selected the GCN4 transcription factor, one of the best-characterized bZIP proteins, which specically binds to the ATF/CREB (50- ATGAcgTCAT-30)orAP1 (50-ATGA(c/g)TCAT-30) consensus sequences. 15 The DNA-binding peptide fragment (br) was chosen based on previous reports that determined the smallest fragment of the GCN4 basic region that preserves the DNA binding attributes of the full protein when engineered as a disulde dimer. 16 Considering the versatility and wide applicability of cysteine alkylations for the selective bioconjugation of peptides and proteins, 17 we selected peptide fragments with a C-terminal cysteine unit. As aryl-containing linking moiety we synthesized product 2, featuring thiol-reactive bromoacetyl units (Fig. 2a). The commercially available precursor 2,6-bis(hydroxymethyl)-p-cresol 1was rst protected as bis-TBS derivative. Aer reaction of the resulting phenol with propargyl bromide to afford 1b, the TBS group was removed and the diol bis-acylated to give the desired dibromide (overall yield of the synthetic process: 22%). The chemoselective coupling between this bis-electrophilic linker and the cysteine-containing peptide brC, was carried out in 100 mM aqueous phosphate buffer (pH 7.8) containing 25% of CH 3 CN, using 2.1 equiv. of the peptide. HPLC-MS analysis of the reaction showed the complete disappearance of the dibromide aer 1.5 h at rt, and the formation of a new major species identied as the expected dimeric peptide (Fig. S10†). The DNA binding of the resulting dimeric construct Pr(brC) 2 was rst analyzed by electrophoretic mobility shiassays (EMSA) under non-denaturing conditions 18 and using SYBR gold for DNA staining. 19 Thus, when a double stranded oligonucleotide containing the target ATF/CREB site (ATF/CREB) was incubated with increasing concentrations of Pr(brC) 2 in Tris– HCl buffer at 20 C, we observed the appearance of a new retarded band consistent with the formation of a specic DNA/ Pr(brC) 2 complex (Fig. 3). The shiof this band is in line with that observed when the same oligonucleotide is incubated with the reference disulde dimer (brGCC) 2 (Fig. 3, lane 6). 14 A control EMSA experiment with an oligonucleotide containing a random sequence does not show retarded bands, thus con- rming that Pr(brC) 2 is a highly selective DNA binder (Fig. S14†). Fluorescence anisotropy titrations using a FAM- Fig. 2 Synthesis of (a) the bis-electrophilic linker 2and (b) the GCN4 basic region dimer Pr(brC) 2 by bis-alkylation with the reactive linker 2. Aba ¼acetamido benzoyl (included as a chromophore to quantify the peptide). Fig. 3 DNA binding of Pr(brC) 2 studied by EMSA. Lanes 1–6: 50 nM target ATF/CREB; lanes 2–5: 250, 500, 750, 1000 nM Pr(brC) 2 ; lane 6: 400 nM (brGCC) 2 . Peptides and dsDNA were mixed in 18 mM Tris–HCl pH 7.5, 90 mM KCl, 1.8 mM MgCl 2 , 1.8 mM EDTA, 9% glycerol, 0.11 mg mL 1 BSA, 2.25% NP-40 for 30 min at 20 C, and loaded into gel. Gel was run on 10% nondenaturing polyacrylamide and 0.5TBE buffer over 40 min, and stained with SyBrGold (5 mLin50mLof1TBE) for 10 min. ATF/CREB (binding site in italics, only one strand is shown): 50- CGG ATGACGTCAT TTTTTTTC-30. © 2022 The Author(s). Published by the Royal Society of Chemistry RSC Adv., 2022, 12,3500–3504 | 3501 Paper RSC Advances Open Access Article. Published on 26 January 2022. Downloaded on 1/27/2022 9:30:43 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online labeled ATF/CREB dsDNA conrmed the high affinity interaction of the peptide dimer Pr(brC) 2 with the ds-oligonucleotide, with an apparent K D of 321 nM at 20 C (Fig. S16†). We next studied the cleavage of Pr(brC) 2 in the absence of DNA. As palladium reagents we selected commercially available complex [Pd(allyl)Cl] 2 (Pd1) and pi-allyl derivative Pd2, both previously shown to mediate depropargylation reactions in water, and even in complex biological settings. 6,20 To our delight, incubation of a solution of Pr(brC) 2 (10 mM) in 20 mM Tris buffer (pH 7.5) with either Pd1 or Pd2 (100 mM) for 3 h at room temperature led to the disappearance of the starting dimer (peak at 18.7 min, see Fig. 4 and S13,†81% conversion aer 2 h). 21 The chromatogram showed a new peak at 17.3 min which was identied as the monomeric fragment 3by MS (Fig. S12†), which is consistent with the cleavage of the linker and the release of the peptide arms (brC) as terminal acids (Fig. 4). Very importantly, the Pd-promoted process can be performed aer formation of the peptide/DNA complex, leading to the expected disassembling. Therefore, when the target DNA (500 nM) was incubated in buffered solution (20 mM Tris buffer pH 7.5, 100 mM KCl) with Pr(brC) 2 (5 mM), and the mixture was treated with either Pd1 or Pd2 (50 mM) for 3 h, we could clearly observe by EMSA the disappearance of most of the retarded band corresponding to the Pr(brC) 2 -DNA complex (band b in Fig. 5, lanes 4–5 and Fig. S15†). A control experiment with the disulde dimer (brGCC) 2 revealed that the DNA binding is retained aer incubation with Pd1 or Pd2 (band a in Fig. 5, lanes 2–3 and Fig. S15†), which conrms that the cleavage/ disassembly sequence only occurs with our responsive peptide dimer. Moreover, in agreement with the EMSA, circular dichroism experiments revealed that addition of 1 equiv. of the dsDNA ATF/CREB to a 5 mM solution of Pr(brC) 2 or (brGCC) 2 promotes a signicant increase in the ellipticity of the negative bands at 208 and 222 nm, consistent with the expected folding of the basic regions into a-helices (Fig. 6). 11,14 Treatment of the solution containing the Pr(brC) 2 –dsDNA complex (5 mM) with Pd1 or Pd2 (50 mM) in 10 mM phosphate buffer (pH 7.5), for 3 h a rt, induced a signicant decrease in ellipticity, as expected for single monomers. 8a,13c In contrast, the same addition to the control mixture containing the (brGCC) 2 peptide did not promote any change. All these results are in total agreement with a Pd-promoted self-immolative cleavage of the propargylic phenol connector, and subsequent release of the two monomeric basic regions (3), which are not capable to efficiently bind to the ATF/CREB sequence. In conclusion, we have designed and developed a new palladium-cleavable linker that can work as temporary dimerizing unit. The utility of the strategy was demonstrated for the construction of functional bZIP dimeric peptides which bind the consensus DNA sequences with high affinity. However, the DNA binding can be efficiently suppressed upon incubation with readily available palladium reagents under mild and physiological conditions. Together with its easy synthetic accessibility, these results support the use of the selfimmolative linker 2as an effective dimerizer that can be readily cleaved in aqueous buffers using transition metal reagents. The methodology, which is complementary to other uncaging strategies based on the use of light, should be Fig. 4 (a) Representation of the Pd-triggered cleavage of the selfimmolative peptide dimer Pr(brC) 2 . (b) HPLC of the self-immolation of Pr(brC) 2 promoted by the Pd1 reagent. Gradient 10 to 50% B over 40 min. Fig. 5 Palladium-promoted cleavage of Pr(brC) 2 studied by EMSA (same conditions as described before). Lanes 1–5: 50 nM target ATF/ CREB; lanes 2–3: 500 nM (brGCC) 2 ; lanes 4–5: 500 nM Pr(brC) 2 . Concentration of Pd1 is, when present, 5 mM. Fig. 6 Circular dichroism of a 5 mM solution of (left) Pr(brC) 2 and (right) (brGCC) 2 in 10 mm phosphate buffer pH 7.5 100 mM of NaCl in the absence of ATF-CREB DNA (dashed line), and after subsequent addition of 1 eq. ATF/CREB DNA (solid line), followed by addition of 10 eq. of Pd1 (B)orPd2 (C). The contributions of the DNA to the CD are subtracted for clarity. All experiments were carried out at 20 C. 3502 |RSC Adv., 2022, 12,3500–3504 © 2022 The Author(s). Published by the Royal Society of Chemistry RSC Advances Paper Open Access Article. Published on 26 January 2022. Downloaded on 1/27/2022 9:30:43 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online extendible to switch offother type of functions that depend on the formation of dimers, as well as for releasing active products from inactive bifunctional precursors. Furthermore, the known ability of the palladium catalyst to promote depropargylation reactions in living systems, 6a suggest that this type of switches can be translated to in vivo settings. Conflicts of interest There are no conicts to declare. Acknowledgements We are thankful for the nancial support from the Xunta de Galicia (Centro singular de investigaci´ on de Galicia acreditaci´ on 2019–2022, ED431G 2019/03) and the European Union (European Regional Development Fund –ERDF). We also thank support given by the Spanish grants PID2019-108624RB-I00, the Xunta de Galicia (grants 2015-CP082, ED431C 2017/19 and 2021-CP054, ED431C-2021/25) and the European Research Council (Advanced Grant No. 340055). MMC and JR thank the MCIN/AEI for their Postdoctoral fellowships (IJCI-2014-19326 and IJC2019-040358-I). Authors would like to thank the use of RIAIDT-USC analytical facilities. Notes and references 1 J. Li and P. R. Chen, Development and application of bond cleavage reactions in bioorthogonal chemistry, Nat. Chem. Biol., 2016, 12, 129–137. 2(a) H. M. Lee, D. R. Larson and D. S. Lawrence, Illuminating the Chemistry of Life: Design, Synthesis, and Applications of “Caged”and Related Photoresponsive Compounds, ACS Chem. Biol., 2009, 4, 409–427; (b)M.I.S ´ anchez, O. V´ azquez, M. E. V´ azquez and J. L. Mascare˜ nas, Lightcontrolled DNA binding of bisbenzamidines, Chem. Commun., 2011, 47, 11107–11109; (c) J. Rodr´ ıguez, J. Mosquera, J. R. Couceiro, M. E. V´ azquez and J. L. Mascare˜ nas, Ruthenation of Non-stacked Guanines in DNA G-Quadruplex Structures: Enhancement of c-MYC Expression, Angew. Chem., Int. Ed., 2016, 55, 15615–15618; (d) M. Mart´ ınez-Calvo, L. Guerrini, J. 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Published by the Royal Society of Chemistry RSC Advances Paper Open Access Article. Published on 26 January 2022. Downloaded on 1/27/2022 9:30:43 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online