Photoswitchable Peptides as Tools for Lightcontrolled Modulation of Epigenetic Protein Complexes

This cumulative thesis consists of two published articles in Chemical Science and ChemBioChem, a submitted manuscript and a published review in Chemical Communications, which provides a detailed overview of photoswitchable peptides capable of modulating biological functions. General aspects as used...

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Bibliographic Details
Main Author: Albert, Lea
Contributors: Vázquez, Olalla (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2020
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Summary:This cumulative thesis consists of two published articles in Chemical Science and ChemBioChem, a submitted manuscript and a published review in Chemical Communications, which provides a detailed overview of photoswitchable peptides capable of modulating biological functions. General aspects as used photoswitches, incorporation techniques and structural precedents are discussed too. Besides the articles, the thesis includes a general overview of epigenetics, which is focused on the methyltransferase MLL1 complex and its inhibitors that target the protein-protein interaction (PPI) MLL1-WDR5. The cumulative part of the thesis is followed by an outlook and conclusions, drawn from the published/submitted manuscripts, and an additional section, which deals with further developments, which were not discussed in the publications. During the last years, we pursued the control of epigenetic states using photoswitchable molecules instead of genetically encoded photoreceptors. For this purpose, we focused on the histone methyltransferase Mixed-Lineage-Leukemia 1 (MLL1) also known as lysine [K]-specific Methyl Transferase 2A (KMT2A), which is a promising target in cancer therapy. Its activity depends on the formation of a protein-protein core complex (MLL1, WDR5, RbBP5, Ash2L, DPY30), where especially the WDR5-MLL1 interaction is of crucial importance. Such PPI is essential to facilitate the MLL1 binding to the RbBP5-Ash2L dimer, needed to maintain the catalytically active conformation of the SET1-domain of MLL1. Based on reported peptides, capable of binding to WDR5, we conceived photoswitchable PPI modulators for altering MLL1 activity. Thus, initially we hypothesized that the incorporation of the classical azobenzene 4-[(4’-aminomethyl)phenyl-azo]benzoic acid (AMPB) into the MLL1-derivative peptide: H2N-SARAEVHLRKS-CONH2 would cause a light-triggered conformational change, which may affect its WDR5 affinity and, thereby, MLL1 activity. Fluorescence polarization (FP)-based experiments revealed that the photoswitchable peptidomimetic H2N-SARA-AMPB-VHLRKS-CONH2 was the most promising candidate of our positional scanning library for the enzymatic activity studies. Hence, this compound displayed the highest affinity window between isomers: Ki (trans) = 1.25 ± 0.36 nM versus Ki (cis) = 6.50 ± 1.4 nM, as well as one order of magnitude tighter binding than the parental MLL1-derivative peptide. Our co-crystal structure (PDB: 5M23) could explain the latter result as a consequence of further stabilization due to an additional hydrogen bond between the first nitrogen of the diazenyl group to the Lys259 as well as van der Waals interactions between the second benzene ring of the AMPB and the Tyr260. Furthermore, our peptidomimetic did not only strongly bind to WDR5 but also inhibited the methylation activity of the MLL1 complex, according to radioactive histone methyltransferase (HMT) assays. Importantly, the difference between isomers was amplified (HMT activity IC50 (trans) = 0.531 ± 0.023 µM; IC50 (cis) = 8.23 ± 0.38 µM), demonstrating for the first time that photoswitchable peptidomimetics can control the activity of histone methyltransferases. The precise control of MLL1 engages a pivotal role in the transcriptional regulation of genes involved in hematopoiesis as H3K4 methylation is associated with transcriptionally active genes. Since MLL1-target genes, such as hox and deptor, are highly overexpressed in MLL-rearranged leukemias, we checked the effect of our peptide on leukemia cell viability. For this purpose, we prepared cell-penetrating versions of the best peptide candidate to explore the modulation of cell growth in leukemia mouse bone marrow cells (MLL-AF9 cells). Cell viability assays verified effective inhibition with a modest, yet clear, difference up to 1.52-fold in the cytotoxic behaviour of isomers (GI50 (trans) = 2.14 ± 0.10 µM, GI50 (cis) = 3.25 ± 0.23 µM). In addition, our most potent inhibitor was tested on its ability to downregulate the expression of deptor via reverse transcriptase real time quantitative PCR (RT-qPCR). These experiments verified that our probe is, indeed, an optoepigenetic inhibitor because it differently downregulated deptor expression levels depending on the photoisomer (relative expression levels of deptor: trans = 0.23 %; cis = 0.36 %, p = 0.008). This proof-of-concept study resulted in an article published in Chemical Science. Once that we demonstrated the potential of photopharmacological inhibition of MLL1 for external epigenetic control, we used this optimized biological system as a platform to improve the photoswitchable peptidomimetics. In particular, we strove for maximizing the difference in biological output between isomers and improving the photochemical properties. Thus, we established the synthesis of two visible-light responsive peptide backbone photoswitches: a Fmoc-protected cyclic azobenzene analogue of 5,6-dihydrodibenzol[c,g][1,2]diazocine (Fmoc-cAzoAA) and a Mtt-ethylenediamine protected tetra-ortho-fluoroazobenzene (Mtt-oF4AzoAA). Apart from avoiding UV irradiation, the oF4Azo switch displayed higher photoconversions than the AMPB derivative (95% cis; 86% trans). However, when they were introduced into the same MLL1-derivative peptide, they did not provide an increased difference in WDR5 binding compared to our former results (H2N-SARA-cAzo-VHLRKS-CONH2 Ki (trans) = 140 ± 35 nM, Ki (cis) = 207 ± 52nM; H2N-SARA-oF4Azo-VHLRKS-CONH2 Ki (trans) = 11.8 ± 1.4 nM, Ki (cis) = 30.8 ± 3.3 nM). Structure-activity relationships deduced from crystallization and theoretical calculations revealed substantial differences in the positioning of the azobenzene moieties. This project was published in ChemBioChem, and highlighted as a front cover. Finally, we combined the visible-light oF4Azo with a structurally-defined cyclic scaffold in an attempt to maximize the biological output. Thus, we designed and synthesized 14 photoswitchable cyclic peptide analogues based on the scaffold of the MLL1 inhibitor: MM-401 ((H3C)2CHCO-NH-c[D-Lys-Arg-Abu-D-Phg]#). Following the same workflow, FP-based assays revealed that the candidate (H3C)2CHCO-NH-c[D-Dab-Arg-Abu-D-Phe-oF4Azo]# displayed an increase of 50% in the WDR5 affinity window between isomers (Ki (trans) = 78.6 ± 3.4 nM, Ki (cis) = 7.99 ± 0.79 nM) compared with our linear AMPB peptide. This confirms that a cyclic constrain leads to greater modulation potential than the lineal arrangement. Unlike the linear AMPB peptide, the cis isomer exhibited both higher WDR5 affinity and MLL1 inhibition activity compared to the trans ones: IC50 (trans) = 6.68 ± 3.1 µM; IC50 (cis) = 0.649 ± 0.3 µM. Crystallization studies, molecular docking (MD) in combination with virtual docking (VD) studies, pull-down assays and hydrogen deuterium exchange (HDX) mass spectrometry (MS) corroborated the increased difference in WDR5 affinity, and its effect on the whole MLL1 complex topology. Via HDX-MS, we discovered that the observed activity loss upon cis-cycle addition was yielded by i.a. allosteric rearrangements of the WDR5-RbBP5 binding interface, leading to the inability of the RbBP5-Ash2L dimer to stabilize the open SET1 conformation, which is required for its activity. Importantly, our cyclic PPI modulators presented improved photochromic properties, behaving as a quasi bistable system. Indeed, the photostationary states were stable for at least five months in darkness. Furthermore, we examined whether the loss of MLL1-enzymatic activity could modulate viability of human leukemia cells. Cell permeabilization via a peptide carrier varied for different cell types, and preliminary tests using the same MLL-AF9 cells as in the Chemical Science, proved that such cells were not suitable. The best results were obtained in MOLM-13 cells, where viability differences of trans: 37 %, cis: 0 % at 200 µM and trans: 57 %, cis: 26 % at 100 µM between isomers were detected. To exclude that the observed toxicity arises from the oF4Azo-moiety itself, the enantiomer of our best oF4Azo-containing cyclic peptide, where the Arg was exchanged by a D-Lys (D-Lys variant), was tested upon its effects on cell viability. Besides, a washing step after cargo transfection was included into the experiment to ensure that the observed effects resulted solely from intracellular mechanisms. Such experiments, with the D-Lys variant as a control proved that the detected toxicity is not originated from the oF4Azo-moeity since low cytotoxicity was observed at 100 µM, being even lower with the active cis isomer (viability: trans: 70 %, cis: 91 %). Also, no interaction of the D-Lys variant with WDR5 was detected by FP-based assays. In contrast, the active cis isomer of our best Arg-containing photoswitchable cyclic candidate caused high toxicity under the same conditions, while the trans isomer retained the same effect (viability: cis: 24 %, trans: 66 %). Ultimately, we tested the in vivo effect of our compound on the modulation of hematopoiesis. We chose the zebrafish developmental model, because MLL1 is conserved in zebrafish, and its knock out represses hematopoiesis. This lack of blood flow is observable by o-dianisidine staining. Gratifyingly, we demonstrated that the cis isomer is capable to affect hematopoiesis in 3 days post fertilization (dpf) zebrafish larvea (o-dianisidine staining: 60% highly disrupted, 40% no staining), which displayed curved body axes, heart edema and a lack of response upon touch. In contrast, 100 % of trans incubated larvea, as well as water and DMSO controls, did not exhibit any of these defects at all. The possibility of visible-light isomerization of our novel cyclic peptide allowed us to study whether trans incubated larvae could be in situ activated. Such experiment proved that the high significant differences between isomers could, indeed, be achieved via in situ irradiation to the cis state (o-dianisidine staining: 3% intact, 30% disrupted, 67%: no staining). All these results together demonstrate the possibility of modulating MLL1-dependent hematopoiesis in vivo using cyclic constraint photoresponsive peptides. This project has recently been submitted.
Physical Description:331 Pages