DEVELOPMENT AND OPTIMIZATION OF SCREENING ASSAYS FOR TARGETED MINIPROTEIN THERAPEUTICS: FOCUS ON KV1.3 AND MMP-2
Szekér Patrik
Molekuláris Orvostudományi Tagozat
Dr. Várnai Péter
SE Elméleti Orvostudományi Központ, Beznák Aladár terem
2025-07-03 14:00:00
Patobiokémia
Dr. Ligeti Erzsébet
Dr. Gyöngyösi Norbert
Dr. Csépányi-Kömi Roland
Dr. Dobó József
Dr. Zelkó Romána
Dr. Németh Tamás
Dr. Róna Gergely
Dr. Ruisanchez Éva
This thesis focuses on the development and optimization of HTS (high-throughput screening) assays for two clinically validated, yet difficult-to-target proteins: MMP-2 and Kv1.3, which are implicated in various cancers and chronic inflammatory diseases, respectively. The objective was to establish scalable and reliable screening platforms to facilitate target characterization and enable the high-throughput discovery of novel miniprotein therapeutics.
For MMP-2, two microbead-based flow cytometry assays were developed to characterize CTX and its variants. The Ig-coated bead binding assay confirmed a dissociation constant (Kd) of 0.62–0.75 µM, while the cobalt-coated bead binding assay revealed additional interactions with NRP1 and, to a lesser extent, with CLC-3 and TIMP-2—supporting a potential multi-target role for CTX. To enable HTS applications, CTX was successfully displayed on phages. Experiments confirmed that phage-bound CTX retained its binding properties, supporting its further optimization for glioblastoma therapy and diagnostic applications.
In the case of Kv1.x channels, a systematic scaffold selection process was implemented to support structured testing of Kv1.x chimeras and to assess their suitability. Four scaffolds were selected, each represented by a specific toxin: Vm24, HgTx1, MTX, and ShK. This strategy enabled a comprehensive evaluation and lays the groundwork for future optimization aimed primarily at selective Kv1.3 inhibition. T-only and T+F KcsA-Kv1.1, Kv1.2, and Kv1.3 chimeras were designed, expressed, and evaluated using microplate-based ELISA assays and patch-clamp measurements. Results indicated that T+F chimeras provided more predictive toxin-binding and blocking profiles, especially for Kv1.2 and Kv1.3, while T-only Kv1.1 chimeras were sufficient for accurate screening purposes.
Following this validation, the optimized chimeras were integrated into a biopanning workflow to isolate Kv1.3-binding sequences from a phage library. This approach resulted in up to a 62.5-fold enrichment of Kv1.3-specific binders within a single selection cycle, confirming the system’s effectiveness for identifying selective inhibitors via directed evolution techniques.
