9 research outputs found
Modulation of the Allosteric Communication between the Polo-Box Domain and the Catalytic Domain in Plk1 by Small Compounds
The Polo-like kinases (Plks) are an evolutionary conserved family of Ser/Thr protein kinases that possess, in addition to the classical kinase domain at the N-terminus, a C-terminal polo-box domain (PBD) that binds to phosphorylated proteins and modulates the kinase activity and its localization. Plk1, which regulates the formation of the mitotic spindle, has emerged as a validated drug target for the treatment of cancer, because it is required for numerous types of cancer cells but not for the cell division in noncancer cells. Here, we employed chemical biology methods to investigate the allosteric communication between the PBD and the catalytic domain of Plk1. We identified small compounds that bind to the catalytic domain and inhibit or enhance the interaction of Plk1 with the phosphorylated peptide PoloBoxtide in vitro. In cells, two new allosteric Plk1 inhibitors affected the proliferation of cancer cells in culture and the cell cycle but had distinct phenotypic effects on spindle formation. Both compounds inhibited Plk1 signaling, indicating that they specifically act on Plk1 in cultured cells.Fil: Raab, Monika. Goethe Universitat Frankfurt; AlemaniaFil: Sanhaji, Mourad. Goethe Universitat Frankfurt; AlemaniaFil: Pietsch, Larissa. German Cancer Research Center; Alemania. Goethe Universitat Frankfurt; AlemaniaFil: BĂ©quignon, Isabelle. Goethe Universitat Frankfurt; AlemaniaFil: Herbrand, Amanda K.. Goethe Universitat Frankfurt; AlemaniaFil: SĂŒĂ, Evelyn. Goethe Universitat Frankfurt; AlemaniaFil: Gande, Santosh L.. German Cancer Research Center; Alemania. Goethe Universitat Frankfurt; AlemaniaFil: Caspar, Birgit. Goethe Universitat Frankfurt; AlemaniaFil: Kudlinzki, Denis. Goethe Universitat Frankfurt; Alemania. German Cancer Research Center; AlemaniaFil: Saxena, Krishna. Goethe Universitat Frankfurt; AlemaniaFil: Sreeramulu, Sridhar. Goethe Universitat Frankfurt; AlemaniaFil: Schwalbe, Harald. Goethe Universitat Frankfurt; Alemania. German Cancer Research Center; AlemaniaFil: Strebhardt, Klaus. Goethe Universitat Frankfurt; Alemania. German Cancer Research Center; AlemaniaFil: Biondi, Ricardo Miguel. German Cancer Research Center; Alemania. Goethe Universitat Frankfurt; Alemania. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Oficina de CoordinaciĂłn Administrativa Parque Centenario. Instituto de InvestigaciĂłn en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; Argentin
Comprehensive Fragment Screening of the SARS-CoV-2 Proteome Explores Novel Chemical Space for Drug Development
12 pags., 4 figs., 3 tabs.SARS-CoV-2 (SCoV2) and its variants of concern pose serious challenges to the public health. The variants increased challenges to vaccines, thus necessitating for development of new intervention strategies including anti-virals. Within the international Covid19-NMR consortium, we have identified binders targeting the RNA genome of SCoV2. We established protocols for the production and NMR characterization of more than 80â% of all SCoV2 proteins. Here, we performed an NMR screening using a fragment library for binding to 25 SCoV2 proteins and identified hits also against previously unexplored SCoV2 proteins. Computational mapping was used to predict binding sites and identify functional moieties (chemotypes) of the ligands occupying these pockets. Striking consensus was observed between NMR-detected binding sites of the main protease and the computational procedure. Our investigation provides novel structural and chemical space for structure-based drug design against the SCoV2 proteome.Work at BMRZ is supported by the state of Hesse. Work in Covid19-NMR
was supported by the Goethe Corona Funds, by the IWBEFRE-program 20007375 of state of Hesse, the DFG
through CRC902: âMolecular Principles of RNA-based regulation.â and through infrastructure funds (project
numbers: 277478796, 277479031, 392682309, 452632086, 70653611) and by European Unionâs Horizon 2020 research and innovation program iNEXT-discovery under grant agreement No 871037. BY-COVID receives funding from the European Unionâs Horizon Europe Research and Innovation Programme under grant agreement number 101046203. âINSPIREDâ (MIS 5002550) project, implemented under the Action âReinforcement of the Research and Innovation Infrastructure,â funded by the Operational
Program âCompetitiveness, Entrepreneurship and Innovationâ (NSRF 2014â2020) and co-financed by Greece and the EU (European Regional Development Fund) and the FP7 REGPOT CT-2011-285950ââSEE-DRUGâ project (purchase of UPATâs 700 MHz NMR equipment). The support of the CERM/CIRMMP center of Instruct-ERIC is gratefully acknowledged. This work has been funded in part by a grant of the Italian Ministry of University and Research (FISR2020IP_02112, ID-COVID) and by Fondazione CR
Firenze. A.S. is supported by the Deutsche Forschungsgemeinschaft [SFB902/B16, SCHL2062/2-1] and the Johanna Quandt Young Academy at Goethe [2019/AS01]. M.H. and C.F. thank SFB902 and the Stiftung Polytechnische Gesellschaft for the Scholarship. L.L. work was supported by the French National Research Agency (ANR, NMR-SCoV2-ORF8), the Fondation de la Recherche MĂ©dicale (FRM, NMR-SCoV2-ORF8), FINOVI and the IR-RMN-THC Fr3050 CNRS. Work at UConn Health was supported by grants from the US National Institutes of Health (R01 GM135592 to B.H., P41 GM111135 and R01 GM123249 to J.C.H.) and the US National Science Foundation (DBI 2030601 to J.C.H.). Latvian Council of Science Grant No. VPP-COVID-2020/1-0014. National Science Foundation EAGER MCB-2031269. This work was supported by the grant Krebsliga KFS-4903-08-2019 and SNF-311030_192646 to J.O. P.G. (ITMP) The EOSC Future project is co-funded by the European Union Horizon Programme call INFRAEOSC-03-2020âGrant Agreement
Number 101017536. Open Access funding enabled and organized by Projekt DEALPeer reviewe
Chemiluminescence Biosensor for the Determination of Cardiac Troponin I (cTnI)
Cardiac vascular diseases, especially acute myocardial infarction (AMI), are one of the leading causes of death worldwide. Therefore cardio-specific biomarkers such as cardiac troponin I (cTnI) play an essential role in the field of diagnostics. In order to enable rapid and accurate measurement of cTnI with the potential of online measurements, a chemiluminescence-based immunosensor is presented as a proof of concept. A flow cell was designed and combined with a sensitive CMOS camera allowing sensitive optical readout. In addition, a microfluidic setup was established, which achieved selective and quasi-online cTnI determination within ten minutes. The sensor was tested with recombinant cTnI in phosphate buffer and demonstrated cTnI measurements in the concentration range of 2â25 ”g/L. With the optimized system, a limit of detection (LoD) of 0.6 ”g/L (23 pmol/L) was achieved. Furthermore, the selectivity of the immunosensor was investigated with other recombinant proteins, such as cTnT, and cTnC, at a level of 16 ”g/L. No cross-reactivity could be observed. Measurements with diluted blood plasma and serum resulted in an LoD of 60 ”g/L (2.4 nmol/L) and 70 ”g/L (2.9 nmol/L), respectively
Comprehensive Fragment Screening of the SARS-CoV-2 Proteome Explores Novel Chemical Space for Drug Development
SARS-CoV-2 (SCoV2) and its variants of concern pose serious challenges to the public health. The variants increased challenges to vaccines, thus necessitating for development of new intervention strategies including anti-virals. Within the international Covid19-NMR consortium, we have identified binders targeting the RNA genome of SCoV2. We established protocols for the production and NMR characterization of more than 80 % of all SCoV2 proteins. Here, we performed an NMR screening using a fragment library for binding to 25 SCoV2 proteins and identified hits also against previously unexplored SCoV2 proteins. Computational mapping was used to predict binding sites and identify functional moieties (chemotypes) of the ligands occupying these pockets. Striking consensus was observed between NMR-detected binding sites of the main protease and the computational procedure. Our investigation provides novel structural and chemical space for structure-based drug design against the SCoV2 proteome.ISSN:1433-7851ISSN:1521-3773ISSN:0570-083
Targeting the main protease (Mpro, nsp5) by growth of fragment scaffolds exploiting structure-based methodologies
The main protease Mpro, nsp5, of SARS-CoV-2 (SCoV2) is one of its most attractive drug targets. Here, we report primary screening data using nuclear magnetic resonance spectroscopy (NMR) of four different libraries and detailed follow-up synthesis on the promising uracil-containing fragment Z604 derived from these libraries. Z604 shows time-dependent binding. Its inhibitory effect is sensitive to reducing conditions. Starting with Z604, we synthesized and characterized 13 compounds designed by fragment growth strategies. Each compound was characterized by NMR and/or activity assays to investigate their interaction with Mpro. These investigations resulted in the four-armed compound 35b that binds directly to Mpro. 35b could be cocrystallized with Mpro revealing its noncovalent binding mode, which fills all four active site subpockets. Herein, we describe the NMR-derived fragment-to-hit pipeline and its application for the development of promising starting points for inhibitors of the main protease of SCoV2
Comprehensive Fragment Screening of the SARSâCoVâ2 Proteome Explores Novel Chemical Space for Drug Development
SARSâCoVâ2 (SCoV2) and its variants of concern pose serious challenges to the public health. The variants increased challenges to vaccines, thus necessitating for development of new intervention strategies including antiâvirals. Within the international Covid19âNMR consortium, we have identified binders targeting the RNA genome of SCoV2. We established protocols for the production and NMR characterization of more than 80 % of all SCoV2 proteins. Here, we performed an NMR screening using a fragment library for binding to 25 SCoV2 proteins and identified hits also against previously unexplored SCoV2 proteins. Computational mapping was used to predict binding sites and identify functional moieties (chemotypes) of the ligands occupying these pockets. Striking consensus was observed between NMRâdetected binding sites of the main protease and the computational procedure. Our investigation provides novel structural and chemical space for structureâbased drug design against the SCoV2 proteome