Yi-Han Chien (1), Judith Röske (2), Kaixuan Zhang (2), Graham P. Marsh (3), Hannah J. Maple (3), Alex Moloney (3), Rolf Hilgenfeld (2,4), Mark Brönstrup (1,5), Katharina Rox (1,5)
(1) Department of Chemical Biology (CBIO), Helmholtz Centre for Infection Research (HZI), Braunschweig, Germany, (2) Institute of Molecular Medicine, University of Lübeck, Lübeck, Germany, (3) Bio-Techne (Tocris), Bristol, United Kingdom, (4) German Center for Infection Research (DZIF), Partner site Lübeck-Borstel-Riems, Germany (5) German Center for Infection Research (DZIF), Partner site Hannover-Braunschweig, Germany
Objectives: Proteolysis-targeting chimeras (PROTAC) are novel chemical concepts taking advantage of the proteasome to degrade target proteins. Compared to PROTACs designed for oncology, which have already entered clinical trials [1], antiviral PROTACs are currently still in an early stage of drug development. Unlike oncological PROTACs, targeting primarily overexpressed endogenous proteins, antiviral PROTACs often target either host proteins, ‘hijacked’ to support viral infection, or viral proteins, presented during infection. To predict the degradation potential of antiviral PROTACs, we adjusted and fine-tuned current modelling frameworks to simulate efficacy of PROTACs compared to conventional small-molecule inhibitors.
Methods: For the modelling framework, we used a hypothetical compound selectively degrading SARS-CoV-2 main protease (Mpro) by employing 13b-K as warhead [2, 3] and pomalidomide as E3-ligase binder [4]. The mechanistic model of PROTAC, published by Haid et al. [5] was deployed to predict the in vitro degradation potential based on the affinity of 13b-K to Mpro and on the affinity of pomalidomide towards cereblon [4]. The feasibility of this hypothetical PROTAC was assessed through parameter adjustments based on various scenarios, incorporating considerations for potential change in affinity as well as the emergence of solubility issues. To get a first preview of potential PK/PD relationships, a PBPK model of the warhead 13b-K was constructed by PK-Sim® as a model for the PK behavior of a 13b-K-based PROTAC using in-house measured ADME and animal experiment data. It was linked with the mechanistic model of PROTAC to predict the in vivo degradation potential.
Results: The model evaluated the degradation efficiency by simulating the phenotypic assay result of the hypothetical compound based on the assay result of its warhead 13b-K. Some assay-specific parameters, including the half-life of Mpro as well as the cereblon concentration were adjusted according to our in-house measurements, and can be consistently maintained as constants when assessing other compounds under comparable conditions. The simulation visualized the difference of a PROTAC from its warhead in phenotypic assay, and identified the concentration intervals specific to each scenario where these differences are expected to be evident. The in vivo degradation potential of the hypothetical compound in lung was illustrated assuming a similar PK behavior as its warhead. It was identified that the degradation effect of a PROTAC often becomes evident and distinguishable from its inhibition effect during the elimination phase.
Conclusions: Our model is a leading attempt to implement a mechanistic PD model of antiviral PROTACs. The proposed modelling framework offers feedback not only on PROTAC structure design, but also on optimizing in vitro assay design by suggesting concentration ranges and on the selection of appropriate cell lines for optimal experimental conditions. Furthermore, it enables an indirect validation on PROTAC degradation efficiency through phenotypic assays when a direct measurement is not possible. This approach enables an initial rough assessment of degradation efficiency using limited affinity data of the compound, potentially minimizing unnecessary experimentation and accelerate the compound screening process. Future studies will focus to link this framework to a viral dynamic model to better anticipate the in vivo PD effect and guide dosing for animal experiments as first milestone for preclinical development.
References:
[1] Békés M, Langley DR, Crews CM. PROTAC targeted protein degraders: the past is prologue. Nature Reviews Drug Discovery. 2022;21(3):181-200.
[2] Zhang L, Lin D, Sun X, Curth U, Drosten C, Sauerhering L, et al. Crystal structure of SARS-CoV-2 main protease provides a basis for design of improved α-ketoamide inhibitors. Science. 2020;368(6489):409-12.
[3] Cooper MS, Zhang L, Ibrahim M, Zhang K, Sun X, Röske J, et al. Diastereomeric Resolution Yields Highly Potent Inhibitor of SARS-CoV-2 Main Protease. J Med Chem. 2022;65(19):13328-42.
[4] Zorba A, Nguyen C, Xu Y, Starr J, Borzilleri K, Smith J, et al. Delineating the role of cooperativity in the design of potent PROTACs for BTK. Proc Natl Acad Sci U S A. 2018;115(31):E7285-e92.
[5] Haid RTU, Reichel A. A Mechanistic Pharmacodynamic Modeling Framework for the Assessment and Optimization of Proteolysis Targeting Chimeras (PROTACs). Pharmaceutics. 2023;15(1).
Reference: PAGE 32 (2024) Abstr 11124 [www.page-meeting.org/?abstract=11124]
Poster: Methodology - Study Design