Chemogenetic profiling reveals PP2A‐independent cytotoxicity of proposed PP2A activators iHAP1 and DT‐061 DOI Creative Commons
Gianmatteo Vit, Joana Duro, Girish Rajendraprasad

et al.

The EMBO Journal, Journal Year: 2022, Volume and Issue: 41(14)

Published: June 13, 2022

Article13 June 2022Open Access Source DataTransparent process Chemogenetic profiling reveals PP2A-independent cytotoxicity of proposed PP2A activators iHAP1 and DT-061 Gianmatteo Vit orcid.org/0000-0002-3111-0769 Novo Nordisk Foundation Center for Protein Research, Faculty Health Medical Sciences, University Copenhagen, Denmark Contribution: Conceptualization, Formal analysis, Supervision, Funding acquisition, Validation, ​Investigation, Visualization, Methodology, Writing - original draft, review & editing Search more papers by this author Joana Duro orcid.org/0000-0002-2608-4420 Data curation, Girish Rajendraprasad orcid.org/0000-0003-2521-3005 Cell Division Cytoskeleton, Danish Cancer Society Research Center, Emil P T Hertz Lya Katrine Kauffeldt Holland Death Metabolism Unit, Autophagy, Recycling Disease (CARD), (DCRC), Melanie Bianca Weisser Brennan C McEwan Department Biochemistry Biology, Geisel School Medicine at Dartmouth College, Hanover, NH, USA Norris Cotton Lebanon, Blanca Lopez-Mendez orcid.org/0000-0001-8541-4904 Paula Sotelo-Parrilla orcid.org/0000-0002-6333-1005 Wellcome Trust Edinburgh, UK A Arockia Jeyaprakash orcid.org/0000-0002-1889-8635 Guillermo Montoya Niels Mailand orcid.org/0000-0002-6623-709X Kenji Maeda Arminja Kettenbach orcid.org/0000-0003-3979-4576 Marin Barisic orcid.org/0000-0001-7587-3867 Cellular Molecular Medicine, Jakob Nilsson Corresponding Author [email protected] orcid.org/0000-0003-4100-1125 Information Vit1,†, Duro1,†, Rajendraprasad2,†, Hertz1, Holland3, Weisser1, McEwan4,5, Lopez-Mendez1, Sotelo-Parrilla6, Jeyaprakash6, Montoya1, Mailand1, Maeda3, Kettenbach4, Barisic2,7 *,1 1Novo 2Cell 3Cell 4Department 5Norris 6Wellcome 7Department † These authors contributed equally to work *Corresponding author. Tel: +45 21328025; E-mail: The EMBO Journal (2022)41:e110611https://doi.org/10.15252/embj.2022110611 PDFDownload PDF article text main figures. Peer ReviewDownload a summary the editorial decision including letters, reviewer comments responses feedback. ToolsAdd favoritesDownload CitationsTrack CitationsPermissions Figures Info Abstract phosphatase 2A (PP2A) is an abundant phosphoprotein that acts as tumor suppressor. For reason, compounds able activate are attractive anticancer agents. have recently been reported selectively stabilize specific PP2A-B56 complexes mediate cell killing. We were unable detect direct effects on activity in biochemical assays composition holoenzymes. Therefore, we undertook genome-wide CRISPR-Cas9 synthetic lethality screens uncover biological pathways affected these compounds. found knockout mitotic regulators lethal with while endoplasmic reticulum (ER) Golgi components DT-061. Indeed showed directly blocks microtubule assembly both vitro vivo thus poison. In contrast, disrupts apparatus ER lipid synthesis associated structures. Our provides insight into perturbed causing cellular toxicity argues cannot be used dissecting biology. Synopsis small-molecule described protein (PP2A). This shows their not mediated PP2A, but instead involves disruption Golgi/ER structures depolymerization, respectively. uncovers potential poison arresting cells prometaphase. No could established. Introduction (PPP) accounts much serine/threonine eukaryotic cells, thereby suppressing multiple oncogenic kinase (Westermarck Hahn, 2008; Kauko Westermarck, 2018; Vainonen et al, 2021). holoenzyme consists catalytic subunit (PP2AC, B isoforms), scaffold (PPP2R1, isoforms) one four regulatory subunits (B55α-δ, B56α-ε, PR72/PR130 or STRN1-4, isoforms each subunit) (Virshup, 2000; 2016; Nilsson, 2019). confer specificity holoenzymes binding substrates substrate specifiers (Cundell 2016). Given suppressed cancers either through overexpression inhibitory proteins mutations components, there interest developing can reactivate cancer treatment Neel, 2020; Several based phenothiazine phenoxazine scaffold, which tricyclic heterocycle include perphenazine (PPZ), DT-061, shown modulate stoichiometry/activity, mediating (Fig 1A) (Gutierrez 2014; Leonard Morita 2020). screen FDA-approved kill T-cell acute lymphoblastic leukemia (T-ALL) antipsychotic drug identified 2014). Through mass spectrometry-based approaches, target was argued PPP2R1A. parallel work, heterocyclic FoxO1 localization reengineered remove central nervous system, making them (Kastrinsky 2015). reengineering led identification several (small-molecule SMAPs), bind PPP2R1A Kd 235 nM, site mapped amino acids 194-198 assays, 20 μM PP2A-B56γ PPP2R1A-PP2AC complex 20–30% (Sangodkar 2017). Figure 1. Perphenazine, no effect Chemical structure perphenazine, iHAP1. Thermogram (top panel) calorimetric titration (400 μM, syringe) (30 cell) (300 (20 25°C corresponding ligand-normalized integrated heats (bottom panel). Experiments performed 5% DMSO. At least two independent experiments performed. Control injection Fig EV1F. dephosphorylation assay using basofilic phosphopeptide purified peptide incubated together indicated. reaction stopped after 15 min, amount phosphate released measured. Mean SD from three technical replicates out shown. MP distribution PP2A-B56α final concentration 50 nM absence (blue) presence (green) 2.5 µM PBS. photometry measurement similar species representative experiment Download figure PowerPoint recent publications, discoveries extended (Leonard al (2020) improved remove, among others, off-target microtubules generate novel compound named (Morita part screening library adding 3 h, followed purification affinity-tagged PP2AC coupled testing. It specifically stabilizes PP2A-B56ε leukemic Kelly causes strong arrest prometaphase selective MYBL2 PP2A-B56ε. mechanism action also investigated approach, they PP2A-B55α results contrast carried structure-function analysis PP2A-B56α-DT-061 analyzed cryo-EM, revealed interacts interface formed all holoenzyme. close very C-terminal flexible tail subunit, residues B56α allow accommodation explaining other B56 isoforms. Here, extensively characterized explored mechanisms chemogenic profiling. argue independently targeting complexes. Results Tricyclic do initially first act As characterization activator contained experimental data confirming binding, important investigate. By isothermal calorimetry (ITC) NMR, any interaction between observed PP2A-B56α/ε reconstituted (Figs 1B EV1A B, Appendix Figs S1–S3). Based result, decided further investigate approaches. did failed reproduce ITC NMR C, EV1C–F S1, S2, S4 S5). next if has stabilizing quantitatively measure solutions. some dissociation prevented addition 1D). Click here expand figure. EV1. A. Titration different preparations determine appropriate use enzymatic assays. Experiment twice concentrations enzyme stated Shown experiment. B–E. model phosphopeptides PPZ, indicated concentrations. (E) BRCA2 WT: P(pS)QKAEITELSTILEESGSQW 2A: P(pS)QKAEITEASTALEESGSQW. experiments. F. Injection μM) buffer (5% DMSO) iHAP 25°C. significant due dilution (control 1B) Collectively, confirm dependent vitro, set impact formation cells. Incubation affect stoichiometries stabilization PP2A-B56α, respectively therefore treated HEK-293T stably expressing myc-tagged 2 30 timeframe sufficient induce clear (see below), immunopurified myc-PP2AC. myc-PP2AC functional it complement RNAi-mediated depletion (Nasa tested Western blotting detected quantitative spectrometry Dataset EV1). PPP2AR1, blot B56ε reveal changes 2C). complementary lysates untreated iHAP1/DT-061-treated microcystin beads endogenous PPP pulled down (Lyons 2018) 2D, EV2). If certain stabilized compound, would unbiased MS interactome analysis. Again, statistically detection B55 (P-value < 0.05, log2 ratio > 1). To explore size exclusion chromatography total extracts HeLa H358 Firstly, little free present fully co-migrated ~ 300 kDa despite 5-fold Secondly, see increase B56α/PPP2R1A peak fractions upon h (Appendix S6 S7). 2. knockdown A–C. stable inducible line (panel B) myc-PP2R1A (C) DMSO, min affinity purified, (A C) (B). (A) Representative repeats (B) triplicate. D. column capture Bound identify components. Analysis conducted E. Doxycyclin-inducible lines subunits. Growth measured 12 days treatment. available online [embj2022110611-sup-0003-SDataFig2.zip] complexes, system (McKinley Cheeseman, induced expression gRNAs B56α, B56δ Cas9, resulted large fraction population becoming full null up EV2A). then monitored viability determined LC50. LC50 values when targets removed CRISPR, arguing unrelated activation 2E). hypothesis increased levels result where YFP-B56α (Wang Ueki previously rescue phenotypes line, than 90% express induction. Indeed, 10-fold without affecting EV2B C). Under same conditions, severely inhibited growth. EV2. structural CRISPR-Cas9-inducible removal doxycycline, Actin loading control. Incucyte growth YFP-B56α. Induced Doxycycline before imaging. DMSO (Ctrl). repeated times mean SD. extract prepared doxycycline-inducible Cells 24 separated Superdex200 column. migration molecular weight markers recombinant untagged top. Licor western PP2A/A, PP2A/C. Venus-B56α PP2A/A below. co-migrating 2–3-fold sample induced. once. (i) superposition crystallographic electron densities maps PDB 2IAE chain a,b,c 3FGA (light pink) cryo-EM map EMD-0510 (gray) threshold 0.25 maps, density protruding center horseshoe-shaped position (closeup, right). fits structures—PDB 6NTS (ii), (iii) 2IAE, (chain a,b,c) (iv) —into respective show

Language: Английский

Dual Inhibition of Vacuolar-ATPase and TMPRSS2 Is Required for Complete Blockade of SARS-CoV-2 Entry into Cells DOI Creative Commons

Simoun Icho,

Edurne Rujas,

Krithika Muthuraman

et al.

Antimicrobial Agents and Chemotherapy, Journal Year: 2022, Volume and Issue: 66(7)

Published: June 15, 2022

An essential step in the infection life cycle of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is proteolytic activation viral spike (S) protein, which enables membrane fusion and entry into host cell. Two distinct classes proteases have been implicated S protein step: cell-surface serine proteases, such as transmembrane protease, (TMPRSS2), endosomal cathepsins, leading to through either route or route, respectively. In cells expressing TMPRSS2, inhibiting using nonspecific cathepsin inhibitors E64d lysosomotropic compounds hydroxychloroquine fails prevent entry, suggesting that unimportant; however, mechanism-based toxicities poor efficacy these confound our understanding importance entry. Here, identify better pharmacological agents elucidate role we profiled a panel molecules identified high-throughput screen inhibit pH and/or maturation different mechanisms. Among three inhibitors, found vacuolar-ATPase macrolide bafilomycin A1 was only agent able potently block without associated cellular toxicity. Using both pseudotyped authentic virus, showed inhibits SARS-CoV-2 absence presence TMPRSS2. Moreover, synergy observed upon combining with Camostat, TMPRSS2 inhibitor, neutralizing TMPRSS2-expressing cells. Overall, this study highlights for provides rationale generation successful intervention strategies against virus combine pathways.

Language: Английский

Citations

26
The green tea catechin EGCG provides proof-of-concept for a pan-coronavirus attachment inhibitor DOI Creative Commons
Emmanuelle V. LeBlanc, Che C. Colpitts

Scientific Reports, Journal Year: 2022, Volume and Issue: 12(1)

Published: July 28, 2022

Abstract The COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has emphasized serious threat to human health posed emerging coronaviruses. Effective broadly-acting antiviral countermeasures are urgently needed prepare for future CoVs, as vaccine development is not compatible with a rapid response newly virus. green tea catechin, epigallocatechin gallate (EGCG), broad-spectrum activity, although its mechanisms against coronavirus (CoV) infection have remained unclear. Here, we show that EGCG prevents and murine CoV blocks entry of lentiviral particles pseudotyped spike proteins from bat or highly pathogenic including SARS-CoV-2 variants concern, in lung epithelial cells. Mechanistically, treatment reduces attachment target cell surfaces interfering cell-surface glycans. Heparan sulfate proteoglycans required factor shown here be important endemic HCoV-OC43 infection. We can compete heparin, heparan analog, virion binding. Our results highlight conserved demonstrate potential pan-coronavirus inhibitors, which may useful protect CoVs.

Language: Английский

Citations

25
Host Kinase CSNK2 is a Target for Inhibition of Pathogenic SARS-like β-Coronaviruses DOI Creative Commons
Xuan Yang, Rebekah J. Dickmander, Armin Bayati

et al.

ACS Chemical Biology, Journal Year: 2022, Volume and Issue: 17(7), P. 1937 - 1950

Published: June 19, 2022

Inhibition of the protein kinase CSNK2 with any 30 specific and selective inhibitors representing different chemotypes, blocked replication pathogenic human, bat, murine β-coronaviruses. The potency in-cell CSNK2A target engagement across set correlated antiviral activity genetic knockdown confirmed essential role holoenzyme in β-coronavirus replication. Spike endocytosis was by inhibition, indicating that due part to a suppression viral entry. inhibition may be viable for development anti-SARS-like drugs.

Language: Английский

Citations

24
Pyronaridine Protects against SARS-CoV-2 Infection in Mouse DOI Open Access
Ana C. Puhl, Giovanni Freitas Gomes, Samara Damasceno

et al.

ACS Infectious Diseases, Journal Year: 2022, Volume and Issue: 8(6), P. 1147 - 1160

Published: May 24, 2022

There are currently relatively few small-molecule antiviral drugs that either approved or emergency-approved for use against severe acute respiratory coronavirus 2 (SARS-CoV-2). One of these is remdesivir, which was originally repurposed from its Ebola. We evaluated three molecules we had previously identified computationally with activity Ebola and Marburg pyronaridine, inhibited the SARS-CoV-2 replication in A549-ACE2 cells. The vivo efficacy pyronaridine has now been assessed a K18-hACE transgenic mouse model COVID-19. Pyronaridine treatment demonstrated statistically significant reduction viral load lungs SARS-CoV-2-infected mice, reducing lung pathology, also associated levels pro-inflammatory cytokines/chemokine cell infiltration. PLpro vitro (IC50 1.8 μM) without any effect on Mpro, indicating possible molecular mechanism involved ability to inhibit replication. have generated several analogs assist understanding structure relationship inhibition. Our results indicate potential therapeutic candidate

Language: Английский

Citations

23
Chemogenetic profiling reveals PP2A‐independent cytotoxicity of proposed PP2A activators iHAP1 and DT‐061 DOI Creative Commons
Gianmatteo Vit, Joana Duro, Girish Rajendraprasad

et al.

The EMBO Journal, Journal Year: 2022, Volume and Issue: 41(14)

Published: June 13, 2022

Article13 June 2022Open Access Source DataTransparent process Chemogenetic profiling reveals PP2A-independent cytotoxicity of proposed PP2A activators iHAP1 and DT-061 Gianmatteo Vit orcid.org/0000-0002-3111-0769 Novo Nordisk Foundation Center for Protein Research, Faculty Health Medical Sciences, University Copenhagen, Denmark Contribution: Conceptualization, Formal analysis, Supervision, Funding acquisition, Validation, ​Investigation, Visualization, Methodology, Writing - original draft, review & editing Search more papers by this author Joana Duro orcid.org/0000-0002-2608-4420 Data curation, Girish Rajendraprasad orcid.org/0000-0003-2521-3005 Cell Division Cytoskeleton, Danish Cancer Society Research Center, Emil P T Hertz Lya Katrine Kauffeldt Holland Death Metabolism Unit, Autophagy, Recycling Disease (CARD), (DCRC), Melanie Bianca Weisser Brennan C McEwan Department Biochemistry Biology, Geisel School Medicine at Dartmouth College, Hanover, NH, USA Norris Cotton Lebanon, Blanca Lopez-Mendez orcid.org/0000-0001-8541-4904 Paula Sotelo-Parrilla orcid.org/0000-0002-6333-1005 Wellcome Trust Edinburgh, UK A Arockia Jeyaprakash orcid.org/0000-0002-1889-8635 Guillermo Montoya Niels Mailand orcid.org/0000-0002-6623-709X Kenji Maeda Arminja Kettenbach orcid.org/0000-0003-3979-4576 Marin Barisic orcid.org/0000-0001-7587-3867 Cellular Molecular Medicine, Jakob Nilsson Corresponding Author [email protected] orcid.org/0000-0003-4100-1125 Information Vit1,†, Duro1,†, Rajendraprasad2,†, Hertz1, Holland3, Weisser1, McEwan4,5, Lopez-Mendez1, Sotelo-Parrilla6, Jeyaprakash6, Montoya1, Mailand1, Maeda3, Kettenbach4, Barisic2,7 *,1 1Novo 2Cell 3Cell 4Department 5Norris 6Wellcome 7Department † These authors contributed equally to work *Corresponding author. Tel: +45 21328025; E-mail: The EMBO Journal (2022)41:e110611https://doi.org/10.15252/embj.2022110611 PDFDownload PDF article text main figures. Peer ReviewDownload a summary the editorial decision including letters, reviewer comments responses feedback. ToolsAdd favoritesDownload CitationsTrack CitationsPermissions Figures Info Abstract phosphatase 2A (PP2A) is an abundant phosphoprotein that acts as tumor suppressor. For reason, compounds able activate are attractive anticancer agents. have recently been reported selectively stabilize specific PP2A-B56 complexes mediate cell killing. We were unable detect direct effects on activity in biochemical assays composition holoenzymes. Therefore, we undertook genome-wide CRISPR-Cas9 synthetic lethality screens uncover biological pathways affected these compounds. found knockout mitotic regulators lethal with while endoplasmic reticulum (ER) Golgi components DT-061. Indeed showed directly blocks microtubule assembly both vitro vivo thus poison. In contrast, disrupts apparatus ER lipid synthesis associated structures. Our provides insight into perturbed causing cellular toxicity argues cannot be used dissecting biology. Synopsis small-molecule described protein (PP2A). This shows their not mediated PP2A, but instead involves disruption Golgi/ER structures depolymerization, respectively. uncovers potential poison arresting cells prometaphase. No could established. Introduction (PPP) accounts much serine/threonine eukaryotic cells, thereby suppressing multiple oncogenic kinase (Westermarck Hahn, 2008; Kauko Westermarck, 2018; Vainonen et al, 2021). holoenzyme consists catalytic subunit (PP2AC, B isoforms), scaffold (PPP2R1, isoforms) one four regulatory subunits (B55α-δ, B56α-ε, PR72/PR130 or STRN1-4, isoforms each subunit) (Virshup, 2000; 2016; Nilsson, 2019). confer specificity holoenzymes binding substrates substrate specifiers (Cundell 2016). Given suppressed cancers either through overexpression inhibitory proteins mutations components, there interest developing can reactivate cancer treatment Neel, 2020; Several based phenothiazine phenoxazine scaffold, which tricyclic heterocycle include perphenazine (PPZ), DT-061, shown modulate stoichiometry/activity, mediating (Fig 1A) (Gutierrez 2014; Leonard Morita 2020). screen FDA-approved kill T-cell acute lymphoblastic leukemia (T-ALL) antipsychotic drug identified 2014). Through mass spectrometry-based approaches, target was argued PPP2R1A. parallel work, heterocyclic FoxO1 localization reengineered remove central nervous system, making them (Kastrinsky 2015). reengineering led identification several (small-molecule SMAPs), bind PPP2R1A Kd 235 nM, site mapped amino acids 194-198 assays, 20 μM PP2A-B56γ PPP2R1A-PP2AC complex 20–30% (Sangodkar 2017). Figure 1. Perphenazine, no effect Chemical structure perphenazine, iHAP1. Thermogram (top panel) calorimetric titration (400 μM, syringe) (30 cell) (300 (20 25°C corresponding ligand-normalized integrated heats (bottom panel). Experiments performed 5% DMSO. At least two independent experiments performed. Control injection Fig EV1F. dephosphorylation assay using basofilic phosphopeptide purified peptide incubated together indicated. reaction stopped after 15 min, amount phosphate released measured. Mean SD from three technical replicates out shown. MP distribution PP2A-B56α final concentration 50 nM absence (blue) presence (green) 2.5 µM PBS. photometry measurement similar species representative experiment Download figure PowerPoint recent publications, discoveries extended (Leonard al (2020) improved remove, among others, off-target microtubules generate novel compound named (Morita part screening library adding 3 h, followed purification affinity-tagged PP2AC coupled testing. It specifically stabilizes PP2A-B56ε leukemic Kelly causes strong arrest prometaphase selective MYBL2 PP2A-B56ε. mechanism action also investigated approach, they PP2A-B55α results contrast carried structure-function analysis PP2A-B56α-DT-061 analyzed cryo-EM, revealed interacts interface formed all holoenzyme. close very C-terminal flexible tail subunit, residues B56α allow accommodation explaining other B56 isoforms. Here, extensively characterized explored mechanisms chemogenic profiling. argue independently targeting complexes. Results Tricyclic do initially first act As characterization activator contained experimental data confirming binding, important investigate. By isothermal calorimetry (ITC) NMR, any interaction between observed PP2A-B56α/ε reconstituted (Figs 1B EV1A B, Appendix Figs S1–S3). Based result, decided further investigate approaches. did failed reproduce ITC NMR C, EV1C–F S1, S2, S4 S5). next if has stabilizing quantitatively measure solutions. some dissociation prevented addition 1D). Click here expand figure. EV1. A. Titration different preparations determine appropriate use enzymatic assays. Experiment twice concentrations enzyme stated Shown experiment. B–E. model phosphopeptides PPZ, indicated concentrations. (E) BRCA2 WT: P(pS)QKAEITELSTILEESGSQW 2A: P(pS)QKAEITEASTALEESGSQW. experiments. F. Injection μM) buffer (5% DMSO) iHAP 25°C. significant due dilution (control 1B) Collectively, confirm dependent vitro, set impact formation cells. Incubation affect stoichiometries stabilization PP2A-B56α, respectively therefore treated HEK-293T stably expressing myc-tagged 2 30 timeframe sufficient induce clear (see below), immunopurified myc-PP2AC. myc-PP2AC functional it complement RNAi-mediated depletion (Nasa tested Western blotting detected quantitative spectrometry Dataset EV1). PPP2AR1, blot B56ε reveal changes 2C). complementary lysates untreated iHAP1/DT-061-treated microcystin beads endogenous PPP pulled down (Lyons 2018) 2D, EV2). If certain stabilized compound, would unbiased MS interactome analysis. Again, statistically detection B55 (P-value < 0.05, log2 ratio > 1). To explore size exclusion chromatography total extracts HeLa H358 Firstly, little free present fully co-migrated ~ 300 kDa despite 5-fold Secondly, see increase B56α/PPP2R1A peak fractions upon h (Appendix S6 S7). 2. knockdown A–C. stable inducible line (panel B) myc-PP2R1A (C) DMSO, min affinity purified, (A C) (B). (A) Representative repeats (B) triplicate. D. column capture Bound identify components. Analysis conducted E. Doxycyclin-inducible lines subunits. Growth measured 12 days treatment. available online [embj2022110611-sup-0003-SDataFig2.zip] complexes, system (McKinley Cheeseman, induced expression gRNAs B56α, B56δ Cas9, resulted large fraction population becoming full null up EV2A). then monitored viability determined LC50. LC50 values when targets removed CRISPR, arguing unrelated activation 2E). hypothesis increased levels result where YFP-B56α (Wang Ueki previously rescue phenotypes line, than 90% express induction. Indeed, 10-fold without affecting EV2B C). Under same conditions, severely inhibited growth. EV2. structural CRISPR-Cas9-inducible removal doxycycline, Actin loading control. Incucyte growth YFP-B56α. Induced Doxycycline before imaging. DMSO (Ctrl). repeated times mean SD. extract prepared doxycycline-inducible Cells 24 separated Superdex200 column. migration molecular weight markers recombinant untagged top. Licor western PP2A/A, PP2A/C. Venus-B56α PP2A/A below. co-migrating 2–3-fold sample induced. once. (i) superposition crystallographic electron densities maps PDB 2IAE chain a,b,c 3FGA (light pink) cryo-EM map EMD-0510 (gray) threshold 0.25 maps, density protruding center horseshoe-shaped position (closeup, right). fits structures—PDB 6NTS (ii), (iii) 2IAE, (chain a,b,c) (iv) —into respective show

Language: Английский

Citations

23