Hacia el entendimiento del control de la expresión génica del regulón clanobacteriano del nitrógeno mediado por el factor de transcripción NtcA y la proteína reguladora PipX

NtcA, es un factor de transcripción cianobacteriano perteneciente a la familia CRP. Es considerado el regulador global del nitrógeno en cianobacterias ya que presenta un regulón muy amplio que incluye muchos genes implicados en el metabolismo de nitrógeno. Se activa por 2-oxoglutarato(2-OG), un indicador del nivel de nitrógeno celular y es coactivado por una proteína llamada PipX, objeto importante de este estudio. A bajo nivel de 2-OG, PipX es secuestrada por la proteína señalizadora PII quedando inaccesible para activar NtcA. Sin embargo, la unión de 2-OG a PII en condiciones de escasez de nitrógeno, libera PipX, la cual queda accesible para coactivar NtcA. Las estructuras de NtcA unido a 2-OG, así como a PipX y 2-OG ya habían sido determinadas previamente en nuestro laboratorio, en esta tesis presentamos la estructura de NtcA unido a su DNA diana y también la estructura del complejo NtcA-PipX-DNA. Estas estructuras aclaran el mecanismo de la especificidad de NtcA por el DNA y muestran que PipX no interacciona con este, sino que estabiliza la conformación activa de NtcA. Así, la unión de PipX a NtcA desplaza el equilibrio entre las formas inactivas del factor de transcripción en favor de su forma competente para unir DNA. Aquí presentamos la estructura de tres formas inactivas de NtcA, sin 2OG unido, demostrando que la conformación inactiva no es específica de especie, sino que existen varias conformaciones inactivas para cada especie. Además, en el complejo PII-PipX, la hélice C-terminal de PipX se encuentra extendida, proporcionando una oportunidad para interaccionar con otras posibles dianas, lo que no podría ocurrir en el complejo NtcA-PipX, donde dicha hélice está siempre flexionada. Nosotros hemos propiciado la determinación de la estructura de PipX por RMN, probando que PipX cuando está libre, presenta su hélice C-terminal flexionada. Recientemente, un regulador génico putativo ha sido identificado como una diana de PipX en el complejo ternario PII-PipX-PlmA. Ya que solo elementos de PipX parecen interaccionar con PlmA en este complejo ternario, parece que PII actúa como un “abridor” de la hélice C-terminal de PipX, promoviendo el secuestro de PlmA en este complejo. Por otra parte, mediante resonancia de plasmón hemos investigado la unión de las proteínas NtcA y CRP a promotores con secuencias consenso canónicas y no canónicas, en ausencia y presencia de sus efectores (2OG y cAMP respectivamente) y/o de la proteína PipX para estudiar qué hace a algunos genes del regulón de NtcA más o menos sensibles a NtcA dependiendo de la presencia o ausencia de 2OG y PipX, y la posible activación cruzada de los promotores de NtcA o CRP, ya que ambos reconocen secuencias de DNA similares. Los resultados muestran que NtcA puede unirse a los promotores dependientes de CRP pero no al revés, además que CRP es incapaz de unirse a su promotor en ausencia de cAMP mientras que NtcA si se une a sus promotores en ausencia de 2OG, aunque con una afinidad menor que en su presencia. También hemos encontrado que la afinidad efectiva de NtcA por el 2OG está muy influenciada por el promotor y que PipX aumenta esta afinidad de forma importante, uniéndose solo a NtcA en presencia de 2OG, apoyando la visión de que PipX estabiliza la conformación activa de NtcA unida a 2OG.NtcA is a cyanobacterial transcriptional regulator from the CRP family of transcription factors. It is considered as the global nitrogen regulator of cyanobacteria because it controls an extense regulon that includes many genes involved in nitrogen metabolism. NtcA is activated by 2-oxoglutarate (2-OG), an indicator of low nitrogen levels in the cell and it is coactivated by protein PipX, an important object of this study. At low 2-OG levels, PipX is sequestered by the PII signaling protein, which makes PipX inaccessible to activate NtcA. However, the binding of 2-OG to PII under nitrogen deprivation conditions releases PipX, rendering it accessible to co-activate NtcA. Structures of NtcA bound to 2-OG in the absence and in the presence of PipX had been previously determined in our laboratory; in this thesis we describe the structure of NtcA bound to its target DNA and also the structure of the NtcA-PipX-DNA complex. These structures clarifies the mechanism of NtcA specificity for its DNA box and also clearly show that PipX does not interact with DNA, but it stabilizes the active conformation of NtcA. Thus, the binding of PipX to NtcA displaces the balance between the multiple inactive forms of the transcription factor in favor of its DNA binding competent form. Here we present also the structure of three inactive forms of NtcA, without 2OG bound, demonstrating that the inactive conformation is not species specific, but that there are several inactive conformations for each species. In addition, in the PII-PipX complex, the C-terminal helix of PipX is extended, providing an opportunity of interaction with other possible targets, which could not occur in the NtcA-PipX complex, where this helix is always flexed. We have propitiated the determination of the PipX structure by NMR, proving that PipX when free, presents its C-terminal helix flexed. Recently, a putative gene regulator has been identified as a target of PipX in the PII-PipX-PlmA ternary complex. Since only PipX elements seem to interact with PlmA in this ternary complex, it seems that PII acts as an "opener" of the C-terminal helix of PipX, promoting the sequestration of PlmA in this complex. On the other hand, by using plasmon resonance technique, we have investigated the binding of NtcA and CRP proteins to promoters with canonical and non-canonical consensus sequences for these transcriptional regulators in the absence and in the presence of their respective effectors (2OG and cAMP respectively) and /or the PipX protein. These experiments were done in order to study what makes some genes of the NtcA regulon more or less sensitive to NtcA depending on the presence or absence of 2OG and PipX, and also to test the possibility of cross-activation betwen NtcA and CRP promoters, due to both transcription factors recognize similar DNA sequences. Our results show that NtcA is able bind to the CRP-dependent promoters but not the other way around. In addition to that, CRP is unable to bind to its own promoter in the absence of cAMP whereas NtcA does bind to its promoters in the absence of 2OG, albeit with less affinity than in his presence. We have also found that the effective affinity of NtcA for 2OG is highly influenced by the promoter and that PipX increases this affinity in an important way, interacting only with NtcA when 2OG is present, supporting the view that PipX stabilizes the active conformation of bound NtcA to 2OG

PipY, a Member of the Conserved COG0325 Family of PLP-Binding Proteins, Expands the Cyanobacterial Nitrogen Regulatory Network

Synechococcus elongatus PCC 7942 is a paradigmatic model organism for nitrogen regulation in cyanobacteria. Expression of genes involved in nitrogen assimilation is positively regulated by the 2-oxoglutarate receptor and global transcriptional regulator NtcA. Maximal activation requires the subsequent binding of the co-activator PipX. PII, a protein found in all three domains of life as an integrator of signals of the nitrogen and carbon balance, binds to PipX to counteract NtcA activity at low 2-oxoglutarate levels. PII-PipX complexes can also bind to the transcriptional regulator PlmA, whose regulon remains unknown. Here we expand the nitrogen regulatory network to PipY, encoded by the bicistronic operon pipXY in S. elongatus. Work with PipY, the cyanobacterial member of the widespread family of COG0325 proteins, confirms the conserved roles in vitamin B6 and amino/keto acid homeostasis and reveals new PLP-related phenotypes, including sensitivity to antibiotics targeting essential PLP-holoenzymes or synthetic lethality with cysK. In addition, the related phenotypes of pipY and pipX mutants are consistent with genetic interactions in the contexts of survival to PLP-targeting antibiotics and transcriptional regulation. We also showed that PipY overexpression increased the length of S. elongatus cells. Taken together, our results support a universal regulatory role for COG0325 proteins, paving the way to a better understanding of these proteins and of their connections with other biological processes.This work was supported by grants from the Valencian (Gerónimo Forteza contract FPA/2015/052 to JL; PrometeoII/2014/029 to VR) and Spanish Governments (BFU2012-33364 and BFU2015-66360-P to AC; BFU2014-58229-P to VR)

Studies on cyanobacterial protein PipY shed light on structure, potential functions, and vitamin B6-dependent epilepsy

The Synechococcus elongatus COG0325 gene pipY functionally interacts with the nitrogen regulatory gene pipX. As a first step toward a molecular understanding of such interactions, we characterized PipY. This 221-residue protein is monomeric and hosts pyridoxal phosphate (PLP), binding it with limited affinity and losing it upon incubation with D-cycloserine. PipY crystal structures with and without PLP reveal a single-domain monomer folded as the TIM barrel of type-III fold PLP enzymes, with PLP highly exposed, fitting a role for PipY in PLP homeostasis. The mobile PLP phosphate-anchoring C-terminal helix might act as a trigger for PLP exchange. Exploiting the universality of COG0325 functions, we used PipY in site-directed mutagenesis studies to shed light on disease causation by epilepsy-associated mutations in the human COG0325 gene PROSC.Supported by grants from the Generalitat Valenciana (PrometeoII/2014/029) and Ministerio de Economía y Competitividad (BFU2014-58229-P to VR; BFU2012-33364 and BFU2015-66360-P to AC; FPI contract to LT) of Spain, and to EC FP7/2007-2013 BioStruct-X (grant agreement no. 283570, proposal 7687) for synchrotron access

The PII-NAGK-PipX-NtcA Regulatory Axis of Cyanobacteria: A Tale of Changing Partners, Allosteric Effectors and Non-covalent Interactions

PII, a homotrimeric very ancient and highly widespread (bacteria, archaea, plants) key sensor-transducer protein, conveys signals of abundance or poorness of carbon, energy and usable nitrogen, converting these signals into changes in the activities of channels, enzymes, or of gene expression. PII sensing is mediated by the PII allosteric effectors ATP, ADP (and, in some organisms, AMP), 2-oxoglutarate (2OG; it reflects carbon abundance and nitrogen scarcity) and, in many plants, L-glutamine. Cyanobacteria have been crucial for clarification of the structural bases of PII function and regulation. They are the subject of this review because the information gathered on them provides an overall structure-based view of a PII regulatory network. Studies on these organisms yielded a first structure of a PII complex with an enzyme, (N-acetyl-Lglutamate kinase, NAGK), deciphering how PII can cause enzyme activation, and how it promotes nitrogen stockpiling as arginine in cyanobacteria and plants. They have also revealed the first clear-cut mechanism by which PII can control gene expression. A small adaptor protein, PipX, is sequestered by PII when nitrogen is abundant and is released when is scarce, swapping partner by binding to the 2OG-activated transcriptional regulator NtcA, co-activating it. The structures of PII-NAGK, PII-PipX, PipX alone, of NtcA in inactive and 2OG-activated forms and as NtcA-2OG-PipX complex, explain structurally PII regulatory functions and reveal the changing shapes and interactions of the T-loops of PII depending on the partner and on the allosteric effectors bound to PII. Cyanobacterial studies have also revealed that in the PII-PipX complex PipX binds an additional transcriptional factor, PlmA, thus possibly expanding PipX roles beyond NtcA-dependency. Further exploration of these roles has revealed a functional interaction of PipX with PipY, a pyridoxal-phosphate (PLP) protein involved in PLP homeostasis whose mutations in the human ortholog cause epilepsy. Knowledge of cellular levels of the different components of this PII-PipX regulatory network and of KD values for some of the complexes provides the basic background for gross modeling of the system at high and low nitrogen abundance. The cyanobacterial network can guide searches for analogous components in other organisms, particularly of PipX functional analogs

Expanding the Cyanobacterial Nitrogen Regulatory Network: The GntR-Like Regulator PlmA Interacts with the PII-PipX Complex

Cyanobacteria, phototrophic organisms that perform oxygenic photosynthesis, perceive nitrogen status by sensing 2-oxoglutarate levels. PII, a widespread signaling protein, senses and transduces nitrogen and energy status to target proteins, regulating metabolism and gene expression. In cyanobacteria, under conditions of low 2-oxoglutarate, PII forms complexes with the enzyme N-acetyl glutamate kinase, increasing arginine biosynthesis, and with PII-interacting protein X (PipX), making PipX unavailable for binding and co-activation of the nitrogen regulator NtcA. Both the PII-PipX complex structure and in vivo functional data suggested that this complex, as such, could have regulatory functions in addition to PipX sequestration. To investigate this possibility we performed yeast three-hybrid screening of genomic libraries from Synechococcus elongatus PCC7942, searching for proteins interacting simultaneously with PII and PipX. The only prey clone found in the search expressed PlmA, a member of the GntR family of transcriptional regulators proven here by gel filtration to be homodimeric. Interactions analyses further confirmed the simultaneous requirement of PII and PipX, and showed that the PlmA contacts involve PipX elements exposed in the PII-PipX complex, specifically the C-terminal helices and one residue of the tudor-like body. In contrast, PII appears not to interact directly with PlmA, possibly being needed indirectly, to induce an extended conformation of the C-terminal helices of PipX and for modulating the surface polarity at the PII-PipX boundary, two elements that appear crucial for PlmA binding. Attempts to inactive plmA confirmed that this gene is essential in S. elongatus. Western blot assays revealed that S. elongatus PlmA, irrespective of the nitrogen regime, is a relatively abundant transcriptional regulator, suggesting the existence of a large PlmA regulon. In silico studies showed that PlmA is universally and exclusively found in cyanobacteria. Based on interaction data, on the relative amounts of the proteins involved in PII-PipX-PlmA complexes, determined in western assays, and on the restrictions imposed by the symmetries of trimeric PII and dimeric PlmA molecules, a structural and regulatory model for PlmA function is discussed in the context of the cyanobacterial nitrogen interaction network.This work was supported by grants BFU2015-66360-P to AC and BFU2014-58229-P to VR from the Spanish Ministry of Economy and Competitivity. AO was the recipient of Grisolia Fellowship from Consellería d'Educació of the Valencian Government and AF-N and LT held FPI fellowships/contracts from Ministry of Economy and Competitivity. JE and VR were supported by grants GV/2014/073 and PrometeoII/2014/029, respectively, from the Consellería d'Educació of the Valencian Government

The PII-NAGK-PipX-NtcA Regulatory Axis of Cyanobacteria: A Tale of Changing Partners, Allosteric Effectors and Non-covalent Interactions

PII, a homotrimeric very ancient and highly widespread (bacteria, archaea, plants) key sensor-transducer protein, conveys signals of abundance or poorness of carbon, energy and usable nitrogen, converting these signals into changes in the activities of channels, enzymes, or of gene expression. PII sensing is mediated by the PII allosteric effectors ATP, ADP (and, in some organisms, AMP), 2-oxoglutarate (2OG; it reflects carbon abundance and nitrogen scarcity) and, in many plants, L-glutamine. Cyanobacteria have been crucial for clarification of the structural bases of PII function and regulation. They are the subject of this review because the information gathered on them provides an overall structure-based view of a PII regulatory network. Studies on these organisms yielded a first structure of a PII complex with an enzyme, (N-acetyl-Lglutamate kinase, NAGK), deciphering how PII can cause enzyme activation, and how it promotes nitrogen stockpiling as arginine in cyanobacteria and plants. They have also revealed the first clear-cut mechanism by which PII can control gene expression. A small adaptor protein, PipX, is sequestered by PII when nitrogen is abundant and is released when is scarce, swapping partner by binding to the 2OG-activated transcriptional regulator NtcA, co-activating it. The structures of PII-NAGK, PII-PipX, PipX alone, of NtcA in inactive and 2OG-activated forms and as NtcA-2OG-PipX complex, explain structurally PII regulatory functions and reveal the changing shapes and interactions of the T-loops of PII depending on the partner and on the allosteric effectors bound to PII. Cyanobacterial studies have also revealed that in the PII-PipX complex PipX binds an additional transcriptional factor, PlmA, thus possibly expanding PipX roles beyond NtcA-dependency. Further exploration of these roles has revealed a functional interaction of PipX with PipY, a pyridoxal-phosphate (PLP) protein involved in PLP homeostasis whose mutations in the human ortholog cause epilepsy. Knowledge of cellular levels of the different components of this PII-PipX regulatory network and of KD values for some of the complexes provides the basic background for gross modeling of the system at high and low nitrogen abundance. The cyanobacterial network can guide searches for analogous components in other organisms, particularly of PipX functional analogs.Supported by grants BFU2014-58229-P and BFU2017-84264-P from the Spanish Government

Expandin Cyanobacterial Nitrogen Regulatory Network: The GntR-Like Regulator PlmA Interacts with the PII-PipX Complex

17 páginas, 8 figuras, 3 tablas. Tiene material suplementario en: http://journal.frontiersin.org/article/10.3389/fmicb.2016.01677/full#supplementary-materialCyanobacteria, phototrophic organisms that perform oxygenic photosynthesis, perceive nitrogen status by sensing 2-oxoglutarate levels. PII, a widespread signaling protein, senses and transduces nitrogen and energy status to target proteins, regulating metabolism and gene expression. In cyanobacteria, under conditions of low 2-oxoglutarate, PII forms complexes with the enzyme N-acetyl glutamate kinase, increasing arginine biosynthesis, and with PII-interacting protein X (PipX), making PipX unavailable for binding and co-activation of the nitrogen regulator NtcA. Both the PII-PipX complex structure and in vivo functional data suggested that this complex, as such, could have regulatory functions in addition to PipX sequestration. To investigate this possibility we performed yeast three-hybrid screening of genomic libraries from Synechococcus elongatus PCC7942, searching for proteins interacting simultaneously with PII and PipX. The only prey clone found in the search expressed PlmA, a member of the GntR family of transcriptional regulators proven here by gel filtration to be homodimeric. Interactions analyses further confirmed the simultaneous requirement of PII and PipX, and showed that the PlmA contacts involve PipX elements exposed in the PII-PipX complex, specifically the C-terminal helices and one residue of the tudor-like body. In contrast, PII appears not to interact directly with PlmA, possibly being needed indirectly, to induce an extended conformation of the C-terminal helices of PipX and for modulating the surface polarity at the PII-PipX boundary, two elements that appear crucial for PlmA binding. Attempts to inactive plmA confirmed that this gene is essential in S. elongatus. Western blot assays revealed that S. elongatus PlmA, irrespective of the nitrogen regime, is a relatively abundant transcriptional regulator, suggesting the existence of a large PlmA regulon. In silico studies showed that PlmA is universally and exclusively found in cyanobacteria. Based on interaction data, on the relative amounts of the proteins involved in PII-PipX-PlmA complexes, determined in western assays, and on the restrictions imposed by the symmetries of trimeric PII and dimeric PlmA molecules, a structural and regulatory model for PlmA function is discussed in the context of the cyanobacterial nitrogen interaction network.This work was supported by grants BFU2015-66360-P to AC and BFU2014-58229-P to VR from the Spanish Ministry of Economy and Competitivity. AO was the recipient of Grisolia Fellowship from Consellería d'Educació of the Valencian Government and AF-N and LT held FPI fellowships/contracts from Ministry of Economy and Competitivity. JE and VR were supported by grants GV/2014/073 and PrometeoII/2014/029, respectively, from the Consellería d'Educació of the Valencian GovernmentPeer reviewe

CryoEM structures of the SARS-CoV-2 spike bound to antivirals

(Póster 63) Background: Single-particle cryoelectron microscopy (cryoEM) has played a key role in the fight against COVID-19. The molecular mechanisms for the action of some of the currently approved drugs targeting the SARS-CoV-2 RNA-dependent RNA polymerase, the fast developments of the current available vaccines and antibody therapies are examples of the impact of the knowledge gained from the cryoEM structures of SARS-CoV-2 proteins in complex with proteins (ACE2 or antibodies/nanobodies) or small compounds. Our aim is to use this technology to understand structurally how certain antiviral compounds and proteins targeting the spike may inhibit viral entry. Methods: 1) Production of wild-type and mutated spike and ACE2 proteins using baculovirus/insect cells. 2) Spike binding kinetics: protein-protein and protein-small compound interactions measured by BLI Biolayer interferometry (BLI) and/or microscale Thermophoresis (MST). 3) Buffer optimization for cryoEM grid preparation of spike variants by thermal shift assays and negative-staining electron microscopy (NSEM). These techniques are also used to adjust the molar ratio of spike:ACE2 and spike:small-compound complexes. 4) Structural characterization by cryoEM. Results: At IBV-CSIC we have created a pipeline for the production and characterization of several spike variants and ACE2 decoys. While this pipeline is described in detail in other oral/poster communications, this communication is centered around one of the pillars within this pipeline; the structural characterization of possible drug candidates bound to the SARS-CoV-2 spike by cryoEM. In this way, we have successfully solved structures of the spike bound to: A) protein inhibitors as ACE2 decoys; B) a small inhibitory compound; C) mixtures of proteins and small-compound (nanobody-heparan derivative) working cooperatively as inhibitors. These protein/drug candidates were previously selected based on the results obtained in our interactomics platform, whereas their concentration and the buffer conditions for cryoEM grids preparation were established based on thermal shift assays and NSEM. Conclusion: CryoEM is a powerful tool to directly visualize the effect caused by a potential drug on a protein target. In a short period of time we have developed this technique in our institute to be applied to the SARS-CoV-2 spike protein, not only to obtain high-resolution structures of SARS- CoV-2 spike variants of concern (see WP4) but also to obtain the structures of complexes of the spike with various inhibitory compounds of very different nature

Use of an interactomics pipeline to assess the potential of new antivirals against SARS-CoV-2

(Póster 80) Background: In late 2019 SARS-CoV-2 infection appeared in China, becoming a pandemic in 2020. The scientific community reacted rapidly, characterizing the viral genome and its encoded proteins, aiming at interfering with viral spreading with vaccines and antivirals. The receptor binding domain (RBD) of the viral spike (S) protein plays a key role in cell entry of the virus. It interacts with the cellular receptor for SARS-CoV-2, the membrane-bound human Angiotensin Converting Ectoenzyme 2 (ACE2). With the goal of monitoring interference with this interaction by potential antiviral drugs, we have set up at the Institute for Biomedicine of Valencia (IBV-CSIC) an interactomics pipeline targeting the initial step of viral entry. Methods: For the production part of the pipeline (pure RBD/Spike variants and soluble ACE2), see parallel poster. These proteins allowed monitoring of the RBD/Spike-ACE2 interaction in presence or absence of potential inhibitors. Thermal shift assays (thermofluor) were used for initial detection of compound binding at different ligand/protein ratios and media conditions (pH, buffers, chaotropic agents). Next, binding affinity and on/off kinetics were characterized using Biolayer interferometry (BLI), Surface plasmon resonance (SPR), Microscale Thermophoresis (MST) and/or Isothermal titration calorimetry (ITC). For protein-protein interactions, we mostly used BLI or SPR, whereas for proteinsmall compound analysis MST was generally best. Protein aggregation-dissociation was monitored by size exclusion chromatography with multiangle light scattering (SEC-MALS). Results: Candidates proven by thermal shift assays to bind to RBD/spike protein without affecting the integrity of these proteins were subjected to quantitative affinity measurements. We successfully demonstrated that BLI, SPR and MST can be used to follow the interactions between SARS-CoV- 2 proteins and the putative drug candidates, as well as to monitor the interference with Spike-Ace2 binding of potential drug candidates. While BLI and SPR displayed reproducible results in the measurement of protein-protein interaction (applied to soluble ACE2 used as a decoy), they were less suitable for measuring the binding of small molecules. The fact that most small compounds were only soluble in organic solvents made difficult to obtain a low signal/noise while using BLI, necessary for the assessment of the binding. We overcame that problem by using MST. After dilution of the compounds to the final experimental concentrations, the technique could detect a significant binding signal enough to calculate binding parameters. MST also allowed to measure the degree of interference that each compound was having on RBD/Spike-ACE2 interaction. The pipeline has been customized and validated with compounds of very different nature provided by different groups belonging to the PTI and other external laboratories, as well as with different Ace2 decoys designed at the IBV. Conclusions: The interactomics platform at the IBV has been used to successfully develop two different antiviral approaches in order to fight COVID-19. It has allowed technical specialization of the staff as well as the development, in a very short period of time, of two ambitious projects. We have demonstrated that we can perform interactomic characterization for challenging projects as well as provide information about binding of antivirals to potential new SARS-CoV-2 variants of concern

Un ataque combinado químico, virológico, biofísico y estructural hace posible la obtención de nuevos inhibidores de entrada celular de SARS-CoV-2 y la caracterización de su mecanismo de inhibición

Resumen del trabajo presentado al 45º Congreso de la Sociedad Española de Bioquímica y Biología Molecular (SEBBM), celebrado en Zaragoza del 5 al 8 de septiembre de 2023.IBV-COVID19 Pipeline: C.Espinosa, N.Gougeard, M.P.Hernández-Sierra, A.Rubio-del-Campo, R.Ruiz-Partida, L.Villamayor.El virus SARS-CoV-2 causa el COVID-19 al infectar las células a través de la interacción de la proteína de su espícula (S) con el receptor celular enzima convertidora de angiotensina 2 (ACE2). Para buscar inhibidores de este paso clave en la infección viral, examinamos una biblioteca interna (IQM-CSIC, Madrid) de compuestos multivalentes derivados de triptófano, primero usando pseudopartículas de Virus de Estomatits Vesicular que expresaban S (I2SysBio, UV y CSIC, Valencia), identificando un compuesto como potente inhibidor de entrada no citotóxico. La optimización química (IQM-CSIC) generó otros dos potentes inhibidores de entrada no citotóxicos que, como 2, también inhibieron la entrada celular de SARS-CoV-2 genuino (I2SysBio). Los estudios con proteínas recombinantes puras (IBV-CSIC, Valencia) usando termofluor y termoforesis de microescala revelaron la unión de estos compuestos a S, y a su dominio de unión al receptor producido separadamente, probando interferencia con la interacción con ACE2. La criomicroscopía electrónica de S (IBV-CSIC), libre o unido al compuesto activo, arrojó luz sobre los mecanismos de inhibición por estos compuestos de la entrada viral a la célula. Esta actividad triinstitucional combinada ha identificado y caracterizado una nueva clase de inhibidores de entrada de SARS-CoV-2 de claro potencial preventivo o terapéutico de COVID-19.ECNextGeneration EUfund 2020/2094 de CSIC/PTI Salud Global; Crue/CSIC/Santander Fondo Supera Covid-19;CSIC-COV19-082; CIBERER-ISCIIICOV20/00437; Covid19-SCI/GValenciana (RG);PID2020- 120322RB-C21 (VR) y PID2020-116880GB-I00 (JLLl) Agenc. Estat Investig.Peer reviewe