Metallopeptides and metalloproteins in chemical biology: from DNA binding to intracellular catalysis

En esta tesis doctoral se describe el diseño, síntesis y aplicación de metalopéptidos y metaloproteínas en diferentes campos, englobados todos ellos dentro del área de la química biológica. En el primer capítulo se describe una nueva generación de péptidos y miniproteínas de unión al ADN, capaces de reconocer sitios diseñados del ADN con alta afinidad y selectividad. El segundo capítulo se centra en un metalopéptido capaz de reconocer una proteína oncogénica y modificar su actividad. En el tercer apartado de la tesis se aborda el uso de un metalopéptido capaz de internalizar en células de mamífero y llevar a cabo reacciones químicas diseñas. Por último, en el capítulo 4, se describe el diseño de unas nuevas metaloproteínas capaces de ensamblarse en el interior celular, estudiando su reactividad para funcionar como metaloenzimas

Assembly of a Ternary Metallopeptide Complex at Specific DNA Sites Mediated by an AT‐Hook Adaptor

The nickel(II)‐mediated self‐assembly of a multimeric DNA binder is described. The binder is composed of two metal‐chelating peptides derived from a bZIP transcription factor (brHis2) and one short AT‐hook domain equipped with two bipyridine ligands (HkBpy2). These peptides reversibly assemble in the presence of NiII ions at selected DNA sequences of 13 base pairsFinancial support from the Spanish grants SAF2016‐76689‐R, RTI2018‐099877‐B‐I00, Orfeo‐cinqa network CTQ2016‐81797‐REDC, the Xunta de Galicia (2015‐CP082, ED431C‐2017/19 and Centro Singular de Investigación de Galicia accreditation 019–2022, ED431G 2019/03), the European Union (European Regional Development Fund—ERDF), and the European Research Council (Advanced Grant No. 340055) are gratefully acknowledged. S.L.‐A. thanks the Spanish MINECO for her FPI fellowship (BES‐2017‐080555); J.R. thanks the Xunta de Galicia for her PhD fellowshipS

Molecular dynamics modelling of the interaction of a synthetic zinc-finger miniprotein with DNA

We report the modelling of the DNA complex of an artificial miniprotein composed of two zinc finger modules and an AT-hook linking peptide. The computational study provides for the first time a structural view of these types of complexes, dissecting interactions that are key to modulate their stability. The relevance of these interactions was validated experimentally. These results confirm the potential of this type of computational approach for studying peptide–DNA complexes and suggest that they could be very useful for the rational design of non-natural, DNA binding miniproteinsThis work has received financial support from Spanish grants (IJC2019-040358-I funded by MCIN/AEI/10.13039/501100011033 to J. R., PID2019-108624RB-I00 funded by MCIN/AEI/10.13039/501100011033 to J. L. M. and RTI2018-096704-B-100 and PID2021-122478NB-I00 to M. O.), the Consellería de Cultura, Educación e Ordenación Universitaria (Grants 2015-CP082, ED431C-2017/19, ED431C-2021/ 25 and ED431G 2019/03: Centro Singular de Investigación de Galicia accreditation 2019–2022 to J. L. M.) and the European Union (European Regional Development Fund-ERDF corresponding to the multiannual financial framework 2014–2020 to J. L. M.). This work was also supported by the BioExcel-2. Centre of Excellence for Computational Biomolecular Research” (823830, M. O.) and the Instituto de Salud Carlos III–Instituto Nacional de Bioinformática (ISCIII PT 17/0009/0007 co-funded by the Fondo Europeo de Desarrollo Regional, M. O.)S

Metal-Dependent DNA Recognition and Cell Internalization of Designed, Basic Peptides

A fragment of the DNA basic region (br) of the GCN4 bZIP transcription factor has been modified to include two His residues at designed i and i+4 positions of its N-terminus. The resulting monomeric peptide (brHis2) does not bind to its consensus target DNA site (5′-GTCAT-3′). However, addition of Pd(en)Cl2 (en, ethylenediamine) promotes a high-affinity interaction with exquisite selectivity for this sequence. The peptide–DNA complex is disassembled by addition of a slight excess of a palladium chelator, and the interaction can be reversibly switched multiple times by playing with controlled amounts of either the metal complex or the chelator. Importantly, while the peptide brHis2 fails to translocate across cell membranes on its own, addition of the palladium reagent induces an efficient cell internalization of this peptide. In short, we report (1) a designed, short peptide that displays highly selective, major groove DNA binding, (2) a reversible, metal-dependent DNA interaction, and (3) a metal-promoted cell internalization of this basic peptideThis work has received financial support from the MINECO (SAF2013-41943-R, SAF2016-76689-R, and CTQ2015-70698-R), the Xunta de Galicia (2015-CP082, ED431C 2017/19, and Centro Singular de Investigación de Galicia Accreditation 2016–2019, ED431G/09), the European Union (European Regional Development Fund, ERDF), and the European Research Council (Advanced Grant No. 340055). Support of COST CM1306 and the orfeo-cinqa network are also acknowledged. J.R. thanks the Xunta de Galicia for a Ph.D. fellowship. We also wish to acknowledge the generous support by the Fundación AECC (IDEAS197VAZQ grant)S

Stimuli-Responsive DNA Binding by Synthetic Systems

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Accounts of chemical research, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see https://doi.org/10.1021/acs.accounts.0c00415Conspectus: DNA is the molecule responsible for the storage and transmission of the genetic information in living organisms. The expression of this information is highly regulated. In eukaryotes, it is achieved mainly at the transcription level thanks to specialized proteins called transcription factors (TFs) that recognize specific DNA sequences, thereby promoting or inhibiting the transcription of particular genes. In many cases, TFs are present in the cell in an inactive form but become active in response to an external signal, which might modify their localization and DNA binding properties or modulate their interactions with the rest of the transcriptional machinery. As a result of the crucial role of TFs, the design of synthetic peptides or miniproteins that can emulate their DNA binding properties and eventually respond to external stimuli is of obvious interest. On the other hand, although the B-form double helix is the most common DNA secondary structure, it is not the only one with an essential biological function. Guanine quadruplexes (GQs) have received considerable attention due to their critical role in the regulation of gene expression, which is usually associated with a change in the GQ conformation. Thus, the development of GQ probes whose properties can be controlled using external signals is also of significant relevance. In this Account, we present a summary of the recent efforts toward the development of stimuli-responsive synthetic DNA binders with a particular emphasis on our own contributions. We first introduce the structure of B and GQ DNAs, and some of the main factors underlying their selective recognition. We then discuss some of the different approaches used for the design of stimulus-mediated DNA binders. We have organized our discussion according to whether the interaction takes place with duplex or guanine quadruplex DNAs, and each section is divided according to the nature of the stimulus (i.e., physical or chemical). Regarding physical stimuli, light (through the incorporation of photolabile protecting groups or photoisomerizable agents) is the most common input for the activation/deactivation of DNA binding events. With respect to chemical signals, the use of metals (through the incorporation of metal-coordinating groups in the DNA binding agent) has allowed the development of a wide range of stimuli-responsive DNA binders. More recently, redox-based systems have also been used to control DNA interactions. This Account ends with a “Conclusions and Outlook” section highlighting some of the general lessons that have been learned and future directions toward further advancing the fieldFinancial support from the Spanish Grants SAF2016-76689-R, RED2018-102417-T, RTI2018-099877-B-I00 the Xunta de Galicia (2015-CP082, ED431B 2018/04, ED431C-2017/19 and Centro Singular de Investigación de Galicia accreditation 2019-2022, ED431G 2019/03), the European Union (European Regional Development Fund - ERDF), and the European Research Council (Advanced Grant No. 340055) are gratefully acknowledgedS

DNA-binding miniproteins based on zinc fingers. Assessment of the interaction using nanopores

Obtaining artificial proteins that mimic the DNA binding properties of natural transcription factors could open new ways of manipulating gene expression at will. In this context it is particularly interesting to develop simple synthetic systems. Inspired by the modularity of natural transcription factors, we have designed synthetic miniproteins that combine the zinc finger module of the transcription factor GAGA and AT-hook peptide domains. These constructs are capable of binding to composite DNA sequences of up to 14 base pairs with high affinity and good selectivity. In particular, we have synthesized three different chimeras and characterized their DNA binding properties by electrophoresis and fluorescence anisotropy. We have also used, for the first time in the study of peptide-based DNA binders, nanopore force spectroscopy to obtain further data on the DNA interaction

Intracellular Reactions Promoted by Bis(histidine) Miniproteins Stapled Using Palladium(II) Complexes

The generation of catalytically active metalloproteins inside living mammalian cells is a major research challenge at the interface between catalysis and cell biology. Herein we demonstrate that basic domains of bZIP transcription factors, mutated to include two histidine residues at i and i+4 positions, react with palladium(II) sources to generate catalytically active, stapled pallado‐miniproteins. The resulting constrained peptides are efficiently internalized into living mammalian cells, where they perform palladium‐promoted depropargylation reactions without cellular fixation. Control experiments confirm the requirement of the peptide scaffolding and the palladium staple for attaining the intracellular reactivityThis work has received financial support from Spanish grants(SAF2016-76689-R, ORFEO-CINQA network CTQ2016-81797-REDC), the Consellería de Cultura, Educación e Ordenación Universitaria (2015-CP082, ED431C-2017/19 and Centro Singular de Investigación de Galicia Accreditation 2019–2022, ED431G 2019/03), the European Union (European Regional Development Fund-ERDF corresponding to the multiannual financial framework 2014–2020), and the European Research Council (Advanced Grant No. 340055). S. L.-A and A. G.-G thank the Ministerio de Educación, Cultura y Deporte for a FPI fellowship (BES-2017-080555) and FPU fellowship (FPU17/00711). C. V. thanks the Ministerio de Economía y Competitividad for the Juan de la Cierva-Formación funding (FJCI-2017-33168)S

Procedimiento de preparación de (-)-englerina A, análogos e intermedios

Procedimiento de preparación de (-)-englerina A, análogos e intermedios. La presente invención se refiere a un procedimiento de obtención del compuesto (-)-englerina A y de compuestos análogos del mismo de fórmula III, donde los grupos R1-R6 tienen el significado descrito en la descripción. Asimismo, la presente invención se refiere a los compuestos análogos mencionados y a su uso en el tratamiento del cáncer, así como a los intermedios de reacción.Peer reviewedConsejo Superior de Investigaciones Científicas (España), Universidad de Santiago de CompostelaA1 Solicitud de patente con informe sobre el estado de la técnic

Controlling oncogenic KRAS signaling pathways with a Palladium-responsive peptide

RAS oncoproteins are molecular switches associated with critical signaling pathways that regulate cell proliferation and differentiation. Mutations in the RAS family, mainly in the KRAS isoform, are responsible for some of the deadliest cancers, which has made this protein a major target in biomedical research. Here we demonstrate that a designed bis-histidine peptide derived from the αH helix of the cofactor SOS1 binds to KRAS with high affinity upon coordination to Pd(II). NMR spectroscopy and MD studies demonstrate that Pd(II) has a nucleating effect that facilitates the access to the bioactive α-helical conformation. The binding can be suppressed by an external metal chelator and recovered again by the addition of more Pd(II), making this system the first switchable KRAS binder, and demonstrates that folding-upon-binding mechanisms can operate in metal-nucleated peptides. In vitro experiments show that the metallopeptide can efficiently internalize into living cells and inhibit the MAPK kinase cascadeS

Selective recognition of A/T-rich DNA 3-way junctions with a three-fold symmetric tripeptide

Non-canonical DNA structures, particularly 3-way junctions (3WJs) that are transiently formed during DNA replication, have recently emerged as promising chemotherapeutic targets. Here, we describe a new approach to target 3WJs that relies on the cooperative and sequence selective recognition of A/T-rich duplex DNA branches by three AT-Hook peptides attached to a three-fold symmetric and fluorogenic 1,3,5-tristyrylbenzene core2023-07-18S