Sebastian Reiner Oehler

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Oehler

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Sebastian Reiner

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0000-0003-2013-7381

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Publications 1 - 8 of 8

DNA-encoded chemical libraries enable the discovery of potent PSMA-ligands with substantially reduced affinity towards the GCPIII anti-target
Lucaroni, Laura; Oehler, Sebastian Reiner; Georgiev, Tony; et al. (2024)
Chemical Science
Prostate-specific membrane antigen (PSMA) is a tumor-associated protein that has been successfully targeted with small organic ligands and monoclonal antibodies. Pluvicto™ is a PSMA-targeted radioligand therapeutic (RLT) recently approved by the FDA for the treatment of metastatic castration-resistant prostate cancer (2022 FDA marketing authorization). Although a large Phase III clinical trial (VISION trial) demonstrated clinical benefits in patients treated with Pluvicto™, the therapeutic window of the drug is narrowed by its undesired accumulation in healthy organs. Glutamate carboxypeptidase III (GCPIII), an enzyme sharing 70% identity with PSMA, may be responsible for the off-target accumulation of PSMA-RLTs in salivary glands and kidneys. In this work, we designed and synthesized affinity and selectivity maturation DNA-encoded chemical libraries (ASM-DELs) comprising 18′284′658 compounds that were screened in parallel against PSMA and GCPIII with the aim to identify potent and selective PSMA ligands for tumor-targeting applications. Compound A70-B104 was isolated as the most potent and selective ligand (K_D of 900 pM for PSMA, K_D of 40 nM for GCPIII). ¹⁷⁷Lu-A70-B104-DOTA, a radiolabeled derivative of compound A70-B104, presented selective accumulation in PSMA-positive cancer lesions (i.e., 7.4% ID g⁻¹, 2 hour time point) after systemic administration in tumor-bearing mice. The results of autoradiography experiments showed that ¹⁷⁷Lu-A70-B104-DOTA selectively binds to PSMA-positive cancer tissues, while negligible binding on human salivary glands was observed.
Discovery of Glutamate Carboxypeptidase III Ligands to Compete the Uptake of [Discovery of Glutamate Carboxypeptidase III Ligands to Compete the Uptake of [¹⁷⁷Lu]Lu-PSMA-617 in Healthy OrgansLu]Lu-PSMA-617 in Healthy Organs
Müller, Marco; Lucaroni, Laura; Favalli, Nicholas; et al. (2024)
Journal of Medicinal Chemistry
Prostate-specific membrane antigen (PSMA)-targeted radio ligand therapeutics (RLTs), such as [¹⁷⁷Lu]Lu-PSMA-617 (Pluvicto), have been shown to accumulate in salivary glands and kidneys, potentially leading to undesired side effects. As unwanted accumulation in normal organs may derive from the cross-reactivity of PSMA ligands to glutamate carboxypeptidase III (GCPIII), it may be convenient to block this interaction with GCPIII-selective ligands. Parallel screening of a DNA-encoded chemical library (DEL) against GCPIII and PSMA allowed the identification of GCPIII binders. Structure–activity relationship (SAR) studies resulted in the identification of nanomolar GCPIII ligands with up to 1000-fold selectivity over PSMA. We studied the ability of GCPIII ligands to counteract the binding of [¹⁷⁷Lu]Lu-PSMA-617 to human salivary glands by autoradiography and could demonstrate a partial radioprotection.
The Influence of Single-Stranded or Double-Stranded DNA Tags on Ligand Binding Affinity in DNA-Encoded Libraries
Bittner, Philipp Peter Rudolf; Gloger, Andreas; Harrison, Julian A.; et al. (2025)
Analytical Chemistry
DNA-encoded libraries have become widely used in drug discovery, and several different setups to link chemical compounds to DNA have been employed in the field, including single-stranded and double-stranded DNA tags as well as a variety of linker chemistries. In our previous study, we observed distinct differences in binding affinities between ligands coupled either to single-stranded or double-stranded DNA; however, the molecular basis for these differences remained unclear. Here, we present a native ion mobility mass spectrometry approach that incorporates gas- and solution-phase activation techniques to systematically investigate these differences, specifically the impact of DNA tags on binding performance in protein–ligand interactions. The results of this study reveal that the binding affinity of DNA-coupled compounds is strongly affected by the DNA setup used. The differences in binding affinities of On-DNA protein–ligand complexes correlated with their differences in thermal stability in solution, with a synergistic binding effect observed exclusively for ligands coupled to single-stranded DNA. This trend is consistent across various protein–ligand systems, which differ in size, binding sites, and ligand structures. Here, we highlight how mass spectrometry can be used to elucidate the impact of different DNA tags on binding dynamics, which is essential for screening and hit validation in DNA-encoded library technology.
Small Organic Carbonic Anhydrase IX Ligands from DNA-Encoded Chemical Libraries for Tumor-Targeted Delivery of Radionuclides
Müller, Marco; Georgiev, Tony; Mock, Jacqueline Caroline; et al. (2025)
Journal of the American Chemical Society
Carbonic anhydrase IX (CAIX) is a membrane protein that is highly expressed in clear cell renal cell carcinoma (ccRCC) and in hypoxic tumors. Being virtually absent in most healthy tissues, CAIX became an attractive target for the selective delivery of diagnostic and therapeutic payloads. Here, we report the discovery and characterization of DNA-encoded chemical library (DEL)-derived CAIX ligands for radionuclide-based imaging applications. Methods: DELs were screened against CAIX and CAII to prioritize hits based on their selectivity and enrichment against CAIX. In vitro characterization of hits was performed by fluorescence polarization (FP), surface plasmon resonance (SPR), and flow cytometry. In vivo biodistribution studies of Lutetium-177 and Gallium-68-radiolabeled compounds were performed in SK-RC-52 tumor-bearing mice. Results: DEL-based CAIX ligands with different affinities and selectivities could be identified. Selectivity and high affinity toward the target correlated with higher tumor-to-organ ratios and improved tumor retention. The best candidate, named OncoCAIX, reached up to similar to 55% injected dose per gram in SK-RC-52 lesions at early time points with very low healthy organ uptake (tumor-to-kidney ratio of >23). Conclusion: OncoCAIX demonstrated rapid and selective tumor uptake, which is a key feature for the development of radionuclide-based imaging agents for early and late-stage ccRCC and hypoxic tumors.
Discovery of high-affinity ligands for prostatic acid phosphatase via DNA-encoded library screening enables targeted cancer therapy
Georgiev, Tony; Migliorini, Francesca; Ciamarone, Andrea; et al. (2025)
Nature Biomedical Engineering
Improving the specificity of prostate cancer treatment requires ligands that bind selectively and with ultra-high affinity to tumour-associated targets absent from healthy tissues. Prostatic acid phosphatase has emerged as an alternative target to prostate-specific membrane antigen, as it is expressed in a broader subset of prostate cancers and is not detected in healthy organs such as the salivary glands and kidneys. Here, to discover selective binders to prostatic acid phosphatase, we constructed two DNA-encoded chemical libraries comprising over 6.7 million small molecules based on proline and phenylalanine scaffolds. Screening against the purified human prostatic acid phosphatase yielded OncoACP3, a small organic ligand with picomolar binding affinity. When radiolabelled with lutetium-177, OncoACP3 selectively accumulated in enzyme-expressing tumours with a long residence time (biological half-life greater than 72 h) and a high tumour-to-blood ratio (>148 at 2 h after administration). Lutetium-177-labelled OncoACP3 cured tumours in mice at low, well-tolerated doses. Its conjugation to the cytotoxic agent monomethyl auristatin E facilitated tumour-selective payload deposition, resulting in potent anti-tumour activity. The modular structure of OncoACP3 supports flexible payload delivery for the targeted treatment of metastatic prostate cancer.
Permutational Encoding Strategy Accelerates HIT Validation from Single-Stranded DNA-Encoded Libraries
Puglioli, Sara; Fabbri, Mosè; Comacchio, Claudia; et al. (2024)
Bioconjugate Chemistry
DNA-Encoded Libraries (DELs) allow the parallel screening of millions of compounds for various applications, including de novo discovery or affinity maturation campaigns. However, library construction and HIT resynthesis can be cumbersome, especially when library members present an unknown stereochemistry. We introduce a permutational encoding strategy suitable for the construction of highly pure single-stranded single-pharmacophore DELs, designed to distinguish isomers at the sequencing level (e.g., stereoisomers, regio-isomers, and peptide sequences). This approach was validated by synthesizing a mock 921,600-member 4-amino-proline single-stranded DEL ("DEL1"). While screening DEL1 against different targets, high-throughput sequencing results showed selective enrichment of the most potent stereoisomers, with enrichment factors that outperform conventional encoding strategies. The versatility of our methodology was additionally validated by encoding 24 scaffolds derived from different permutations of the amino acid sequence of a previously described cyclic peptide targeting Fibroblast Activation Protein (FAP-2286). The resulting library ("DEL2") was interrogated against human FAP, showing selective enrichment of five cyclic peptides. We observed a direct correlation between enrichment factors and on-DNA binding affinities. The presented encoding methodology accelerates drug discovery by facilitating library synthesis and streamlining HIT resynthesis while enhancing enrichment factors at the DEL sequencing level. This facilitates the identification of HIT candidates prior to medicinal chemistry and affinity maturation campaigns.
Phenylalanine-Based DNA-Encoded Chemical Libraries for the Discovery of Potent and Selective Small Organic Ligands Against Markers of Cancer and Immune Cells
Migliorini, Francesca; Ciamarone, Andrea; Dakhel Plaza, Sheila; et al. (2025)
Advanced Science
DNA-encoded chemical libraries (DELs) are powerful tools for drug discovery, enabling the high-throughput screening of vast libraries of small molecules against target proteins of pharmaceutical interest. Here, the synthesis of two new DELs, named FM-DEL1 and FM-DEL2, including 7′710 and 5′697’690 compounds, respectively is described. These libraries are constructed by installing one or two sets of building blocks on a phenylalanine central scaffold. FM-DELs are screened against markers of prostate cancer, and renal cell carcinoma, and against an immunological target expressed on the surface of natural killer cells. Highly potent and selective binders with affinity constants in the nanomolar range are obtained from DEL screenings against those targets. Small-molecule ligands against tumor-associated antigens are used to develop small-molecule radiopharmaceuticals that selectively accumulate at cancer sites after systemic administration.
Native Mass Spectrometry Facilitates Hit Validation in DNA-Encoded Library Technology
Bittner, Philipp Peter Rudolf; Gloger, Andreas; Keller, Michelle; et al. (2025)
Angewandte Chemie. International Edition
DNA-encoded Library (DEL) Technology has become a workhorse of drug development, is widely employed in an industrial and academic setting, and an increasing number of drugs developed by DEL technology have entered clinical stage development. While up to billions of compounds can be screened simultaneously in affinity-based selections, the validation and characterization of individual hits discovered from DEL selections is a substantial bottleneck since it can be cumbersome and time-consuming. Here, we describe the use of native mass spectrometry for the speedy hit validation of On-DNA compounds. Through the preservation of noncovalent interactions in the gas phase, complexes of proteins and their respective On-DNA ligands can be analyzed without further need of labeling or immobilization. By utilizing the workflow described in this work, we were able to reliably rank affinities of various purified On-DNA binders or demonstrate binding of On-DNA compounds from unpurified mixtures, mitigating the need for tedious purification steps. Additionally, the methodology described here can offer valuable insight on which moiety of a binding molecule contributes the most to binding, facilitating subsequent medicinal chemistry efforts for lead expansion.

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Sebastian Reiner Oehler

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