ACE2, the Receptor that Enables the Infection by SARS‐CoV‐2: Biochemistry, Structure, Allostery and Evaluation of the Potential Development of ACE2 Modulators

Angiotensin Converting Enzyme 2 (ACE2) is the human receptor that interacts with the Spike protein of coronaviruses, including the one that produced the 2020 coronavirus pandemic (COVID-19). Thus, ACE2 is a potential target for drugs that disrupt the interaction of human cells with SARS-CoV-2 to abolish infection. There is also interest on drugs that inhibit or activate ACE2, i.e. for cardiovascular disorders or colitis. Compounds binding at alternative sites could allosterically affect the interaction with Spike protein. We here review biochemical, chemical biology and structural information on ACE2, including the recent cryoEM structures of full length ACE2. We conclude that ACE2 is very dynamic and that allosteric drugs may be developed to target ACE2. At the time of the 2020 pandemic, we suggest that available ACE2 inhibitors or activators in advanced development should be tested for their ability to allosterically displace the interaction between ACE2 and the Spike protein.Fil: Gross, Lissy Zoe Florens. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaFil: Sacerdoti, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaFil: Piiper, Albrecht. Goethe Universitat Frankfurt; AlemaniaFil: Zeuzem, Stefan. Goethe Universitat Frankfurt; AlemaniaFil: Leroux, Alejandro Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaFil: Biondi, Ricardo Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; Argentin

Modulation of the substrate specificity of the kinase PDK1 by distinct conformations of the full-length protein

The activation of at least 23 different mammalian kinases requires the phosphorylation of their hydrophobic motifs by the kinase PDK1. A linker connects the phosphoinositide-binding PH domain to the catalytic domain, which contains a docking site for substrates called the PIF pocket. Here, we used a chemical biology approach to show that PDK1 existed in equilibrium between at least three distinct conformations with differing substrate specificities. The inositol polyphosphate derivative HYG8 bound to the PH domain and disrupted PDK1 dimerization by stabilizing a monomeric conformation in which the PH domain associated with the catalytic domain and the PIF pocket was accessible. In the absence of lipids, HYG8 potently inhibited the phosphorylation of Akt (also termed PKB) but did not affect the intrinsic activity of PDK1 or the phosphorylation of SGK, which requires docking to the PIF pocket. In contrast, the small molecule valsartan bound to the PIF pocket and stabilized a second distinct monomeric conformation. Our study reveals dynamic conformations of full-length PDK1 in which the location of the linker and the PH domain relative to the catalytic domain determines the selective phosphorylation of PDK1 substrates. The study further suggests new approaches for the design of drugs to selectively modulate signaling downstream of PDK1

A Chemical Biology Approach to Understand The Regulation Of Full-Length Phosphoinositide-Dependent Protein Kinase 1 (Pdk1)

Phosphoinositide-dependent protein kinase 1 (PDK1) is a master AGC kinase that phosphorylates at least other 23 AGC kinases, being PKB/Aktthe most relevant substrate downstream of PI3-kinase, important for growth and cell survival and a drug target for cancer treatment. Over the years, our laboratory studied and characterized in detail the catalytic domain of PDK1, as well as the selective activation of substrates such as SGK or S6K, which in order to be phosphorylated require a docking interaction with a hydrophobic site in PDK1 termed the PIF-Pocket. However, this is not the case of Akt/PKB, since it can be activated in a PIF-Pocket independent way. On the other hand, up to date little is known about the mechanistic and structural details of PDK1 full length. Therefore, we are using an interdisciplinary approach to understand how the full length protein is regulated and how this regulation mechanism can be manipulated to specifically inhibit the activation of PKB/Akt. As a result of a medium-scale screening of small compounds we carried out using AlphaScreen technology, we validated a series of small compounds "hits" that modulate PDK1 structure by interaction at different sites on PDK1. We performed hydrogen/deuterium exchange (HDX) experimentsand crystallization screenings to understand the structure of full length PDK1 and how it can be modulated with small compounds. We here present a series of results obtained using HDX on full length PDK1 and the crystal structure of the catalytic domain of PDK1 bound to a small compound identified in our screening. We also present our in vitro studies to understand the oligomerization of PDK1 visualizing single particles using STORM fluorescence microscopy. Finally, we integrate our data to present an updated model on the molecular mechanism of regulation of PDK1.Fil: Gross, Lissy Zoe Florens. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaFil: Sacerdoti, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaFil: Leroux, Alejandro Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaFil: Rinaldi, Jimena Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Capellari, M. V.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias "Elizabeth Jares Erijman"; ArgentinaFil: Aramendia, Pedro Francisco. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias "Elizabeth Jares Erijman"; ArgentinaFil: Ghode, A.. National University; SingapurFil: Anand, G. S.. National University; SingapurFil: Klinke, Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Biondi, Ricardo Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaLV Reunión Científica anual SAIB y XIV congreso de PABMBSaltaArgentinaSociedad Argentina de Investigación Bioquímica y Biología MolecularPan-American Association For Biochemistry And Molecular Biolog

Preservation of protective capacity of hyperimmune anti-Stx2 bovine colostrum against enterohemorrhagic Escherichia coli O157:H7 pathogenicity after pasteurization and spray-drying processes

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a major etiologic agent that causes bloody diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (HUS). Shiga toxin (Stx) is the main virulence factor of EHEC responsible for the progression to HUS. Although many laboratories have made efforts to develop an effective treatment for Stx-mediated HUS, a specific therapy has not been found yet. Human consumption of bovine colostrum is known to have therapeutic effects against several gastrointestinal infections because of the peptide and proteins (including antibodies) with direct antimicrobial and endotoxin-neutralizing effects contained in this fluid. We have previously demonstrated that colostrum from Stx type 2 (Stx2)-immunized pregnant cows effectively prevents Stx2 cytotoxicity and EHEC O157:H7 pathogenicity. In this study we evaluated the preservation of the protective properties of hyperimmune colostrum against Stx2 (HIC-Stx2) after pasteurization and spray-drying processes by performing in vitro and in vivo assays. Our results showed that reconstituted HIC-Stx2 colostrum after pasteurization at 60°C for 60 min and spray-dried under optimized conditions preserved specific IgG that successfully neutralized Stx2 cytotoxicity on Vero cells. Furthermore, this pasteurized/dehydrated and reconstituted HIC-Stx2 preserved the protective capacity against EHEC infection in a weaned mice model. The consumption of hyperimmune HIC-Stx2 bovine colostrum could be effective for HUS prevention in humans as well as in EHEC control in calves. However, further studies need to be done to consider its use for controlling EHEC infections.Instituto de VirologíaFil: Garimano, Nicolás. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay. Departamento de Ciencias Fisiológicas. Laboratorio de Fisiopatogenia; ArgentinaFil: Garimano, Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Diaz Vergara, Ladislao Iván. Universidad Nacional de Villa María. Centro de Investigaciones y Transferencia de Villa María; ArgentinaFil: Diaz Vergara, Ladislao Iván. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Kim, A. D. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay. Departamento de Ciencias Fisiológicas. Laboratorio de Fisiopatogenia; ArgentinaFil: Kim, A. D. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Badin, Emiliano Emanuel. Universidad Nacional de Villa María. Centro de Investigaciones y Transferencia de Villa María; ArgentinaFil: Badin, Emiliano Emanuel. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Sodero, Sonia. Universidad Nacional de Villa María. Centro de Investigaciones y Transferencia de Villa María; ArgentinaFil: Sodero, Sonia. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Bernal, Alan Mauro. Academia Nacional de Medicina. Instituto de Medicina Experimental. Laboratorio de Patogénesis e Inmunología de Procesos Infecciosos; ArgentinaFil: Bernal, Alan Mauro. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Gonzalez, Diego. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; ArgentinaFil: Amaral, María Marta. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay. Departamento de Ciencias Fisiológicas. Laboratorio de Fisiopatogenia; ArgentinaFil: Amaral, María Marta. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Lespinard, Alejandro R. Universidad Nacional de Villa María. Centro de Investigaciones y Transferencia de Villa María; ArgentinaFil: Lespinard, Alejandro R. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Porporatto, Carina. Universidad Nacional de Villa María. Centro de Investigaciones y Transferencia de Villa María; ArgentinaFil: Porporatto, Carina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Montenegro, Mariana A. Universidad Nacional de Villa María. Centro de Investigaciones y Transferencia de Villa María; ArgentinaFil: Montenegro, Mariana A. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Palermo, Marina Sandra. Academia Nacional de Medicina. Instituto de Medicina Experimental. Laboratorio de Patogénesis e Inmunología de Procesos Infecciosos; ArgentinaFil: Palermo, Marina Sandra. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Larzabal, Mariano. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular (IABIMO); ArgentinaFil: Larzabal, Mariano. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Cataldi, Angel Adrian. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular (IABIMO); ArgentinaFil: Cataldi, Angel Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Ibarra, Cristina Adriana. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay. Departamento de Ciencias Fisiológicas. Laboratorio de Fisiopatogenia; ArgentinaFil: Ibarra, Cristina Adriana. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Sacerdoti, Flavia. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay. Departamento de Ciencias Fisiológicas. Laboratorio de Fisiopatogenia; ArgentinaFil: Sacerdoti, Flavia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Modulation of the substrate specificity of the kinase PDK1 by distinct conformations of the full-length protein

The activation of at least 23 different mammalian kinases requires the phosphorylation of their hydrophobic motifs by the kinase PDK1. A linker connects the phosphoinositide-binding PH domain to the catalytic domain, which contains a docking site for substrates called the PIF pocket. Here, we used a chemical biology approach to show that PDK1 existed in equilibrium between at least three distinct conformations with differing substrate specificities. The inositol polyphosphate derivative HYG8 bound to the PH domain and disrupted PDK1 dimerization by stabilizing a monomeric conformation in which the PH domain associated with the catalytic domain and the PIF pocket was accessible. In the absence of lipids, HYG8 potently inhibited the phosphorylation of Akt (also termed PKB) but did not affect the intrinsic activity of PDK1 or the phosphorylation of SGK, which requires docking to the PIF pocket. In contrast, the small-molecule valsartan bound to the PIF pocket and stabilized a second distinct monomeric conformation. Our study reveals dynamic conformations of full-length PDK1 in which the location of the linker and the PH domain relative to the catalytic domain determines the selective phosphorylation of PDK1 substrates. The study further suggests new approaches for the design of drugs to selectively modulate signaling downstream of PDK1

Measurement of the W±ZW^{\pm}Z boson pair-production cross section in pppp collisions at s=13\sqrt{s}=13 TeV with the ATLAS Detector

The production of $W^{\pm}Z$ events in proton--proton collisions at a centre-of-mass energy of 13 TeV is measured with the ATLAS detector at the LHC. The collected data correspond to an integrated luminosity of 3.2 fb$^{-1}$. The $W^{\pm}Z$ candidates are reconstructed using leptonic decays of the gauge bosons into electrons or muons. The measured inclusive cross section in the detector fiducial region for leptonic decay modes is $\sigma_{W^\pm Z \rightarrow \ell^{'} \nu \ell \ell}^{\textrm{fid.}} = 63.2 \pm 3.2$ (stat.) $\pm 2.6$ (sys.) $\pm 1.5$ (lumi.) fb. In comparison, the next-to-leading-order Standard Model prediction is $53.4^{+3.6}_{-2.8}$ fb. The extrapolation of the measurement from the fiducial to the total phase space yields $\sigma_{W^{\pm}Z}^{\textrm{tot.}} = 50.6 \pm 2.6$ (stat.) $\pm 2.0$ (sys.) $\pm 0.9$ (th.) $\pm 1.2$ (lumi.) pb, in agreement with a recent next-to-next-to-leading-order calculation of $48.2^{+1.1}_{-1.0}$ pb. The cross section as a function of jet multiplicity is also measured, together with the charge-dependent $W^+Z$ and $W^-Z$ cross sections and their ratio

Search for Scalar Diphoton Resonances in the Mass Range 65−60065-600 GeV with the ATLAS Detector in pppp Collision Data at s\sqrt{s} = 8 TeVTeV

A search for scalar particles decaying via narrow resonances into two photons in the mass range 65–600 GeV is performed using $20.3\text{}\text{}{\mathrm{fb}}^{-1}$ of $\sqrt{s}=8\text{}\text{}\mathrm{TeV}$ $pp$ collision data collected with the ATLAS detector at the Large Hadron Collider. The recently discovered Higgs boson is treated as a background. No significant evidence for an additional signal is observed. The results are presented as limits at the 95% confidence level on the production cross section of a scalar boson times branching ratio into two photons, in a fiducial volume where the reconstruction efficiency is approximately independent of the event topology. The upper limits set extend over a considerably wider mass range than previous searches

Search for Higgs and ZZ Boson Decays to J/ψγJ/\psi\gamma and Υ(nS)γ\Upsilon(nS)\gamma with the ATLAS Detector

A search for the decays of the Higgs and $Z$ bosons to $J/\psi\gamma$ and $\Upsilon(nS)\gamma$ ($n=1,2,3$) is performed with $pp$ collision data samples corresponding to integrated luminosities of up to $20.3\mathrm{fb}^{-1}$ collected at $\sqrt{s}=8\mathrm{TeV}$ with the ATLAS detector at the CERN Large Hadron Collider. No significant excess of events is observed above expected backgrounds and 95% CL upper limits are placed on the branching fractions. In the $J/\psi\gamma$ final state the limits are $1.5\times10^{-3}$ and $2.6\times10^{-6}$ for the Higgs and $Z$ bosons, respectively, while in the $\Upsilon(1S,2S,3S)\,\gamma$ final states the limits are $(1.3,1.9,1.3)\times10^{-3}$ and $(3.4,6.5,5.4)\times10^{-6}$, respectively

Measurements of the Total and Differential Higgs Boson Production Cross Sections Combining the H??????? and H???ZZ*???4??? Decay Channels at s\sqrt{s}=8??????TeV with the ATLAS Detector

Measurements of the total and differential cross sections of Higgs boson production are performed using 20.3~fb$^{-1}$ of $pp$ collisions produced by the Large Hadron Collider at a center-of-mass energy of $\sqrt{s} = 8$ TeV and recorded by the ATLAS detector. Cross sections are obtained from measured $H \rightarrow \gamma \gamma$ and $H \rightarrow ZZ ^{*}\rightarrow 4\ell$ event yields, which are combined accounting for detector efficiencies, fiducial acceptances and branching fractions. Differential cross sections are reported as a function of Higgs boson transverse momentum, Higgs boson rapidity, number of jets in the event, and transverse momentum of the leading jet. The total production cross section is determined to be $\sigma_{pp \to H} = 33.0 \pm 5.3 \, ({\rm stat}) \pm 1.6 \, ({\rm sys}) \mathrm{pb}$. The measurements are compared to state-of-the-art predictions.Measurements of the total and differential cross sections of Higgs boson production are performed using 20.3  fb-1 of pp collisions produced by the Large Hadron Collider at a center-of-mass energy of s=8  TeV and recorded by the ATLAS detector. Cross sections are obtained from measured H→γγ and H→ZZ*→4ℓ event yields, which are combined accounting for detector efficiencies, fiducial acceptances, and branching fractions. Differential cross sections are reported as a function of Higgs boson transverse momentum, Higgs boson rapidity, number of jets in the event, and transverse momentum of the leading jet. The total production cross section is determined to be σpp→H=33.0±5.3 (stat)±1.6 (syst)  pb. The measurements are compared to state-of-the-art predictions.Measurements of the total and differential cross sections of Higgs boson production are performed using 20.3 fb$^{-1}$ of $pp$ collisions produced by the Large Hadron Collider at a center-of-mass energy of $\sqrt{s} = 8$ TeV and recorded by the ATLAS detector. Cross sections are obtained from measured $H \rightarrow \gamma \gamma$ and $H \rightarrow ZZ ^{*}\rightarrow 4\ell$ event yields, which are combined accounting for detector efficiencies, fiducial acceptances and branching fractions. Differential cross sections are reported as a function of Higgs boson transverse momentum, Higgs boson rapidity, number of jets in the event, and transverse momentum of the leading jet. The total production cross section is determined to be $\sigma_{pp \to H} = 33.0 \pm 5.3 \, ({\rm stat}) \pm 1.6 \, ({\rm sys}) \mathrm{pb}$. The measurements are compared to state-of-the-art predictions