Project Details
Projekt
Target - Oriented Screening of a Cytochalasan Compound Library
Applicant
Privatdozent Dr. Carsten Zeilinger
Subject Area
Analytical Chemistry
Biological and Biomimetic Chemistry
Metabolism, Biochemistry and Genetics of Microorganisms
Biological and Biomimetic Chemistry
Metabolism, Biochemistry and Genetics of Microorganisms
Term
since 2021
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 438841444
The importance of protein microarrays as useful tools for biopharmaceutical research is of increasing interest. These micro-spot arrays hold a broad potential for elementary biological discoveries and also for drug development. A substance library of isolated or synthesized cytochalasan derivatives is provided via CytoLabs. The aim is to identify affinities of the compounds for actin in the cellular cell framework protein or to determine other possible cytochalasan targets. This can help to distinguish actin binding activity from other activities. The data obtained are used to optimize in vitro structure-activity relationships (SAR) for the design of better substances or to understand potentially different effects of the broad spectrum of cytochalasin structure variants. In addition, compounds are identified that target biofilm production in prokaryotes such as S. aureus. From preliminary work, the binding of fluorescence-labeled cytochalasan to F-actin and the tumor suppressor protein TP53 was selectively investigated on a microarray. These candidates as well as other protein targets such as heat shock proteins, ion channels and others become tested by the binding and displacement test of fluorescence-labeled substances on target proteins under parallelized test conditions. Furthermore, the binding assay is used to analyze the binding affinities of orthologous organisms from human, fungal, but also bacterial ones like MreB and the influence of the modification at the binding site of the actins. Important binding areas are the binding site for cytochalasin (CBS) and the ATP binding site. Mutations in the cytochalasin binding site (CBS) or ATP-binding site will be generated to affect affinity for compounds or ligands. Another strategy is to search the proteome database for actins with sequence variations in the ATP binding site or CBS. The sequence and structural alignment of actins show that M. grisea actin has some variations in the CBS, and these variations in CBS can hinder or reduce the binding of self-produced cytochalasan. This will be examined more in detail on recombinantly synthesized actin from M. grisea. After that, variations identified in the CBS in other candidates and identified via a proteome database search are transferred to human actin and tested for cytochalasan and / or ATP binding and displacement activity. If these positions affect cytochalasan binding activity, extracts from identified organism candidates are tested for displacement activity. This strategy could help to predict the producer of cytochalasan or related drugs from the proteome database.
DFG Programme
Research Units
