Abstract B147: From bioactive small molecule to an identified protein target: A new method combining stochastic photomodification with a synthetic antibody mimetic
Authors: Blazkova Kristyna , Šimon Petr , Knedlík Tomáš , Dvořáková Petra , Březinová Anna , Kostka Libor , Šubr Vladimír , Konvalinka Jan , Šácha Pavel 
Published: February 2019

The renaissance of phenotypic screening leads to the discovery of biologically active small molecules resulting in a high need to quickly and efficiently identify their protein target. This is most commonly done by a preparation of an affinity probe for the target pull-down, which requires time-consuming structure-activity relationship studies to identify proper position for an attachment of an affinity anchor with necessary linker. We set out to circumvent this step altogether and prepare stochastic conjugates that would enable fast target identification without prior structural information or testing of suitable linker attachment position. We suggest that they could be used also for the identification of unknown targets of phenotypically active compounds as well as off-targets of established active compounds. To test the new approach, we selected several model protein targets – glutamate carboxypeptidase II (GCPII, also known as prostate specific membrane antigen, PSMA) expressed in prostate cancer, fibroblast activation protein (FAP) in tumor stroma, and aspartic protease family, specifically pepsin, cathepsin D and HIV protease. For each, we stochastically modified their corresponding known biologically active small-molecule inhibitors using a linker with a photoactivatable diazirine group. Resulting reaction mixture contained various inhibitor isomers, whose biologic activity was not compromised by the position of the linker attachment as well as those where it was. Afterwards, the reaction mixtures were conjugated to N-(2-hydroxypropyl)methacrylamide copolymers (called iBodies) carrying biotin and a fluorophore, which were used as fully synthetic antibody mimetics. In all cases, stochastic polymer conjugates were able to pull-down the corresponding target protein from cell lysates. For the GCPII conjugate, we then analyzed the pulled-down proteins by mass spectrometry, which clearly identified GCPII as the target protein of the conjugate, even with no prior knowledge needed. For GCPII and FAP, which are cell surface receptors, those same conjugates were able to visualize the proteins on cells both in confocal microscopy and flow cytometry. On a model case of GCPII we decided to identify the position of the “productive” linker attachment so that it would allow rational synthesis for future use. Using HPLC, we separated the reaction mixture into several fractions, each enriched in a specific linker attachment position. As expected, the fractions differed in binding properties compared to the whole mixture. The position of the linker attachment was determined by mass spectrometry for selected active and inactive fractions. The identified linker positions and activity of the compounds corresponded to the known mode of binding of the small molecule into the active site of GCPII. Polymer conjugates prepared from the most favorable fraction had improved binding properties. This “improved” conjugate allowed more efficient isolation and subsequent identification of GCPII by mass spectrometry. These results show that stochastic modification of bioactive small molecule combined with the avidity effect on iBodies allowed us to avoid lengthy structure-activity-relationship studies and enabled identification of protein target and its visualization using a single conjugate. This technology might contribute to fast and facile identification of protein targets of phenotypically active small molecules. This work was supported by Grant No. GA16-02938S from the Grant Agency of the Czech Republic, InterBioMed Project LO 1302 from the Ministry of Education of the Czech Republic, and by the Charles University, project GA UK No. 1510-243-250045.

Citation Format: Kristyna Blazkova, Petr Šimon, Tomáš Knedlík, Petra Dvořáková, Anna Březinová, Libor Kostka, Vladimír Šubr, Jan Konvalinka, Pavel Šácha. From bioactive small molecule to an identified protein target: A new method combining stochastic photomodification with a synthetic antibody mimetic [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B147.