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

The structural role of SARS-CoV-2 genetic background in the emergence and success of spike mutations: The case of the spike A222V mutation

The S:A222V point mutation, within the G clade, was characteristic of the 20E (EU1) SARS-CoV-2 variant identified in Spain in early summer 2020. This mutation has since reappeared in the Delta subvariant AY.4.2, raising questions about its specific effect on viral infection. We report combined serological, functional, structural and computational studies characterizing the impact of this mutation. Our results reveal that S:A222V promotes an increased RBD opening and slightly increases ACE2 binding as compared to the parent S:D614G clade. Finally, S:A222V does not reduce sera neutralization capacity, suggesting it does not affect vaccine effectiveness

C-2 Thiophenyl Tryptophan Trimers Inhibit Cellular Entry of SARS-CoV-2 through Interaction with the Viral Spike (S) Protein

26 páginas, 6 figuras, 2 tablas.Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19, by infecting cells via the interaction of its spike protein (S) with the primary cell receptor angiotensin-converting enzyme (ACE2). To search for inhibitors of this key step in viral infection, we screened an in-house library of multivalent tryptophan derivatives. Using VSV-S pseudoparticles, we identified compound 2 as a potent entry inhibitor lacking cellular toxicity. Chemical optimization of 2 rendered compounds 63 and 65, which also potently inhibited genuine SARS-CoV-2 cell entry. Thermofluor and microscale thermophoresis studies revealed their binding to S and to its isolated receptor binding domain (RBD), interfering with the interaction with ACE2. High-resolution cryoelectron microscopy structure of S, free or bound to 2, shed light on cell entry inhibition mechanisms by these compounds. Overall, this work identifies and characterizes a new class of SARS-CoV-2 entry inhibitors with clear potential for preventing and/or fighting COVID-19.Funding for this project was provided by grants from the European Commission NextGenerationEU fund (EU 2020/2094), through CSIC’s Global Health Platform (PTI Salud Global), Crue-CSIC-Santander Fondo Supera Covid-19, and CSIC grant (CSIC-COV19-082) to R.G., M.-J-P.-P., V.R., J.B., A.M., and J.-L.L. and CIBERER, Instituto de Salud Carlos III (COV20/00437) to V.R., J.B., A.M., and J.-L.L. In addition, this work was funded by grant (Covid_19-SCI) from the Generalitat Valenciana y Conselleria de Innovación, Universidades, Ciencia y Sociedad digital to R.G., and by grants PID2020-120322RB-C21 and PID2020-116880GB-I00 from the Agencia Estatal de Investigación of the Spanish Government to V.R. and J.-L.L., respectively.Peer reviewe

The structural role of SARS-CoV-2 genetic background in the emergence and success of spike mutations: the case of the spike A222V mutation

The S:A222V point mutation, within the G clade, was characteristic of the 20E (EU1) SARS-CoV-2 variant identified in Spain in early summer 2020. This mutation has now reappeared in the Delta subvariant AY.4.2, raising questions about its specific effect on viral infection. We report combined serological, functional, structural and computational studies characterizing the impact of this mutation. Our results reveal that S:A222V promotes an increased RBD opening and slightly increases ACE2 binding as compared to the parent S:D614G clade. Finally, S:A222V does not reduce sera neutralization capacity, suggesting it does not affect vaccine effectiveness.This research work was supported by the European Commission–NextGenerationEU through the CSIC Global Health Platform. Additionally, authors would like to acknowledge economic support from the Spanish Ministry of Science and Innovation through Grants: PID2019-104757RB-I00 funded by MCIN/AEI/ 10.13039/501100011033, RTI2018-094399-A-I00, and “ERDF A way of making Europe”, by the “European Union”, Grant SEV 2017-0712 funded by MCIN/AEI /10.13039/501100011033, the “Comunidad Autónoma de Madrid" through Grant: S2017/BMD3817, and the European Union (EU) and Horizon 2020 through grants: Marie-Curie Fellowship EnLaCES (MSCA IF 2020, Proposal: 101024130) (to JK), HighResCells (ERC - 2018 - SyG, Proposal: 810057), and iNEXT-Discovery (Proposal: 871037). AM, VR, JB and JLL are funded by CIBERER-ISCIII (proposal: COV20/00437), Fondo Supera COVID-19 (proposal: CSICCOVID19-082), Banco Santander (Proposal: BlockAce), and CSIC PTI Salud Global (Proposal: 202080E110). VR is funded by the Spanish Ministry of Science and Innovation through Grant PID2020-120322RB-C21. IC is funded by project PID2019-104477RB-100, Fondo COVID COV20/00140 and ERC CoG 101001038. MC is funded by the RyC program from the Spanish Ministry of Science and Innovation, the Generalitat Valenciana (SEJI/2019/011).N

New findings with the IBV decoy for cell entry inhibition of SARS-CoV-2, and unique structural data for soluble dimeric ACE2 bound to the viral S trimer

Resumen del trabajo presentado a las III Jornadas Científicas PTI+ Salud Global, celebradas en el Centro de Ciencias Humanas y Sociales (CCHS), CSIC (Madrid) del 20 al 22 de noviembre de 2023.[Background] The SARS-CoV-2 spike protein (S) mediates the interaction of the virus with cellular membrane receptor (angiotensin-converting enzyme 2, ACE2). In previous PTI meetings, we reported heterologous production in vitro of the ACE2 extracellular domains modified by site-directed mutagenesis to increase its affinity for the S protein, to enable it to be used as viral entry inhibitor (decoy) by competing with the membrane-bound cellular receptor. We now test the value of these decoys for: 1) binding to S variants that emerged during the evolution of the pandemic in viral lineages of concern; and 2) inhibiting experimental cellular infection by pseudotyped virus expressing these S variants. Cellular syncytia formation has been described in several organs as a manifestation of severe COVID-19, and likely has pathogenic impact. To test further our decoys’ effectiveness, we studied their impact on cellular syncytia formation within an experimental in vitro cell culture model. Searching for effective decoys, we produced monomeric and dimeric ACE2 proteins, depending on the respective absence/presence of the extracellular collectrin domain. Interestingly, there are no reported structures of dimeric soluble ACE2 bound to the S protein. After extensive knowledge-guided trial-and-error, we succeeded in visualizing by cryo-electron microscopy (cryoEM) this interaction (~7-Å-resolution), and in understanding the challenges inherent in determining such a complex structural organization.[Methods] 1) Recombinant production and purification of the monomeric or dimeric ACE2, their decoys the receptor binding domain (RBD) and the S protein variants of interest. We used baculovirus/insect cells to produce ACE2s and RBDs, and human Expi293F cells for the S proteins. 2) Biolayer interferometry for assessing protein-protein interactions; 3) Use of a model system for monitoring viral cellular infection and its inhibition by decoys. We used a pseudotyped engineered vesicular stomatitis virus expressing and exposing at its surface the desired S protein variant, to infect appropriate SARS-CoV-2-susceptible mammalian cells; 4) Single-particle cryoEM; 5) Syncytia formation testing using an engineered cultured cell system in which heterologous surface expression of the S protein in one cell type induces syncytium formation in other cells expressing membrane-bound ACE2.[Results] Our decoys proved highly effective in preventing cellular infection by pseudotyped virus expressing the S proteins of different SARS-CoV-2 variants of concern. Biophysical results have validated the maintained interaction between the decoy and the various S protein variants. When introduced into the cellular model system for syncytia formation, the decoys proved capable of decreasing such formation. Puzzlingly, the monomeric decoy was more effective than the dimeric one. The cryoEM images unveiled an ACE2 dimer configuration, where the subunits, resembling the previously reported monomer, were oriented at an angle of >60º, in which the vortex was the interlinked collectrin domains. Both catalytic domains engage with a single RBD of one subunit from different S trimers. The formation of a network at high stoichiometries of both components poses a challenge for structure determination by cryoEM.[Conclusions] Unlike therapeutic antibodies, which proved ineffective on variants not initially used for their production, our decoys should be effective in preventing infection by all widely widespread SARS-CoV-2 variants.Peer reviewe

The role of SARS-CoV-2 genetic background in the emergence and success of spike mutations: the case of the spike A222V mutation

Resumen del trabajo presentado a las II Jornadas Científicas PTI + Salud Global, celebradas los días 5 y 6 de octubre de 2022 en el Auditorio Santiago Grisolía de Valencia (España).Peer reviewe

CIBERER: Spanish national network for research on rare diseases: A highly productive collaborative initiative

13 páginas,1 figura, 3 tablas, 1 apéndice. Se extraen los autores pertenecientes a The CIBERER network que trabajan en Centros del CSIC del Appendix ACIBER (Center for Biomedical Network Research; Centro de Investigación Biomédica En Red) is a public national consortium created in 2006 under the umbrella of the Spanish National Institute of Health Carlos III (ISCIII). This innovative research structure comprises 11 different specific areas dedicated to the main public health priorities in the National Health System. CIBERER, the thematic area of CIBER focused on rare diseases (RDs) currently consists of 75 research groups belonging to universities, research centers, and hospitals of the entire country. CIBERER's mission is to be a center prioritizing and favoring collaboration and cooperation between biomedical and clinical research groups, with special emphasis on the aspects of genetic, molecular, biochemical, and cellular research of RDs. This research is the basis for providing new tools for the diagnosis and therapy of low-prevalence diseases, in line with the International Rare Diseases Research Consortium (IRDiRC) objectives, thus favoring translational research between the scientific environment of the laboratory and the clinical setting of health centers. In this article, we intend to review CIBERER's 15-year journey and summarize the main results obtained in terms of internationalization, scientific production, contributions toward the discovery of new therapies and novel genes associated to diseases, cooperation with patients' associations and many other topics related to RD research.This study has been funded by Instituto de Salud Carlos III (ISCIII) and Spanish Ministry of Science and InnovationPeer reviewe

Deciphering the mechanism by which 1-pyrrolin-5-carboxylate synthetase defects associate with dominant and recessive human pathologies

Póster original presentado al XXXVIII Congreso de la Sociedad Española de Bioquímica y Biología Molecular (SEBBM), celebrado en Valencia, 7-10 de septiembre de 2015Δ1-Pyrrolin-5-carboxylate synthetase (P5CS) is a bifunctional enzyme that catalyzesthe first two steps of ornithine/proline biosynthesis in plants and animals. It is composed of an N-terminal glutamate-5-kinase (G5K) domain that uses ATP to phosphorylate the γ-carboxylate of glutamate, and of a C-terminal L-glutamyl-5-phosphate reductase (G5PR) domain that reductively dephosphorylates L-glutamyl-5-phosphate to L-glutamate-5-semialdehyde. This compound spontaneously cyclizes to Δ1-pyrrolin-5-carboxylate, thus being a precursor of both ornithine and proline. Human P5CS deficiency was associated with a fasting hyperammonemia syndrome with cataracts and vomiting. Then the syndrome was expanded to include a cutis laxa phenotype and more recently it has been associated with spastic paraplegia of dominant or recessive inheritance. Using our baculovirus/insect cell system for producing pure recombinant human P5CS, we introduced dominant and recessive mutations in the enzyme by site-directed mutagenesis, assaying enzyme activity. All the mutations, irrespectively of the type of inheritance they gave, inactivated the catalytic domain of P5CS where they mapped. Gel filtration and modelling evidence strongly suggests that the mutations giving dominant inheritance appear to cause their effects by a negative dominant mechanism. In particular, they are located on the surface of the two domains, in places that in E. coli G5K (a monofuntional tetrameric enzyme) are used in interactions with adjacent domains. Therefore, these dominant mutations may disturb the hexameric enzyme architecture, preventing potential "in vivo" channelling of L-glutamyl-5-phosphate from the G5K domain to a G5PR domain from an adjacent subunit.BFU2014-58229-P(MINECO).Peer reviewe

N-carbamoylglutamate-responsive carbamoyl phosphate synthetase 1 (CPS1) deficiency: A patient with a novel CPS1 mutation and an experimental study on the mutation's effects

9 páginas, 3 figurasN-carbamoyl-l-glutamate (NCG), the N-acetyl-l-glutamate analogue used to treat N-acetylglutamate synthase deficiency, has been proposed as potential therapy of carbamoyl phosphate synthetase 1 deficiency (CPS1D). Previous findings in five CPS1D patients suggest that NCG-responsiveness could be mutation-specific. We report on a patient with CPS1D, homozygous for the novel p.(Pro1211Arg) CPS1 mutation, who presented at 9 days of life with hyperammonemic coma which was successfully treated with emergency measures. He remained metabolically stable on merely oral NCG, arginine, and modest protein restriction. Ammonia scavengers were only added after poor dietary compliance following solid food intake at age 1 year. The patient received a liver transplantation at 3.9 years of age, having normal cognitive, motor, and quality of life scores despite repeated but successfully treated episodes of hyperammonemia. Studies using recombinantly produced mutant CPS1 confirmed the partial nature of the CPS1D triggered by the p.(Pro1211Arg) mutation. This mutation decreased the solubility and yield of CPS1 as expected for increased tendency to misfold, and reduced the thermal stability, maximum specific activity (V max; ~2-fold reduction), and apparent affinity (~5-fold reduction) for ATP of the purified enzyme. By increasing the saturation of the NAG site in vivo, NCG could stabilize CPS1 and minimize the decrease in the effective affinity of the enzyme for ATP. These observations, together with prior experience, support the ascertainment of clinical responsiveness to NCG in CPS1 deficient patients, particularly when decreased stability or lowered affinity for NAG of the mutant enzyme are suspected or proven.Grants to V.R. from the Fundación Inocente Inocente2018 (Spain) and BFU2017-84264-P of MINECO-SpanishGovernment. N.G. and B.B. had contracts from CIBERER-ISCIII and Fundación Inocente Inocente. Orphan Europefunded editorial assistance.Peer reviewe

Patología molecular y cristalografía de rayos X arrojan nueva luz sobre estructura-función de la carbamil fosfato sintetasa 1 humana (CPS1) (G01 35 P 027)

Trabajo presentado en el 44º Congreso de la SEBBM, celebrado en Málaga (España) del 06 al 09 de septiembre de 2022.(G01 35 P 027) La CPS1 es la entrada-interruptor del ciclo de la urea, operada por su activador esencial N-acetil-L-glutamato (NAG). Este ciclo es clave para la detoxificación del amonio derivado del catabolismo de las proteínas. Nuestras dos estructuras cristalinas de CPS1 humana sin ligandos o unidas a 2ADP, K+, Mg2+ y NAG, demostraron (De Cima et al. Sci Reports2015) su composición por 6 dominios globulares, dos de ellos catalíticos y homólogos, otro de activación por NAG y un cuarto de integración, quedando por determinar la función de sus dos dominios N-terminales (~400 aas.), reminiscentes de la subunidad menor de las CPSs anabólicas bacterianas. Hemos abordado la determinación de dicha función mediante mutagénesis dirigida, usando los datos genéticos de pacientes con déficit de CPS1 como atajo para elegir qué mutaciones introducir. En 30 mutantes humanos de estos dominios, producidos en y purificados de un sistema de baculovirus/células de insecto, hemos realizado ensayos de estabilidad, de actividad enzimática, y determinado las constantes cinéticas para los sustratos y el NAG. Los resultados indican un importante papel estabilizador de los dominios N-terminales, explicando su conservación en CPS1 a pesar de que han perdido la función ancestral glutaminasa de la subunidad menor de las CPSs bacterianas. Las estructuras ya determinadas, representativas de ambos extremos del proceso de activación de la CPS1, junto con una nueva estructura presentada aquí para una forma intermedia de activación pre-unión de NAG, fundamentan estructuralmente dicho papel estabilizador, a la vez que arrojan luz sobre el inicio del proceso de activación.Financiado por el Proyecto CIVP-20A6610 (Ciencias de la Vida) de la Fundación Ramón Areces