Biology subjects

Reidenbach, A. G.

Publications and source records attributed to Reidenbach, A. G..

6 recordsLinked to original sources

bioRxiv2026

Phenotypic screening for small molecules that lower PrP in cultured cells

PrP lowering is a validated therapeutic hypothesis in prion disease. To identify small molecules that reduce PrP levels, we performed phenotypic screening in cultured cells. To prioritize PrP specificity in our primary screen, we generated mouse N2a cells stably expressing GFP and used high content imaging analysis to select compounds that lowered PrP without affecting GFP signal or cell viability. Screening a curated library of 3,492 compounds with annotated mechanisms of action identified two small molecules, EYH (PubChem CID: 71678945) and LCZ (PubChem CID: 24970350), that selectively and dose-dependently lowered PrP. Proteomics on whole cell lysates identified PrP as the #1 or #2 most potently downregulated out of 8,722 proteins detected. Both compounds minimally affected Prnp mRNA, reduced expression of exogenously transfected PrP, and remained potent in non-dividing primary cells, consistent with a post-translational mechanism. Co-treatment with the proteasome inhibitor MG132 yielded accumulation of unglycosylated PrP, demonstrating proteasome clearance of PrP. However, both compounds showed limited or no activity in human cell lines, and failed to reduce PrP in vivo after 14 days of treatment. These findings highlight the challenges associated with mechanism-agnostic phenotypic screening for PrP-lowering compounds and support prioritizing compounds with known mechanisms of action.

neuroscience↗

bioRxiv2024

PrP turnover in vivo and the time to effect of prion disease therapeutics

PrP lowering is effective against prion disease in animal models and is being tested clinically. Therapies in the current pipeline lower PrP production, leaving pre-existing PrP to be cleared according to its own half-life. We hypothesized that PrPs half-life may be a rate-limiting factor for the time to effect of PrP-lowering drugs, and one reason why late treatment of prion-infected mice is not as effective as early treatment. Using isotopically labeled diet with targeted mass spectrometry, as well as antisense oligonucleotide treatment followed by timed PrP measurement, we estimate a half-life of 5-6 days for PrP in the brain. PrP turnover is not affected by over-or under-expression. Mouse PrP and human PrP have similar turnover rates measured in wild-type or humanized knock-in mice. CSF PrP appears to mirror brain PrP in real time in rats. PrP in the colon is readily quantifiable and has a half-life just slightly shorter than in brain. An under-expressed pathogenic mutant PrP, corresponding to D178N in humans, exhibits an accelerated turnover rate. Our data may inform the design of both preclinical and clinical studies of PrP-lowering drugs. Author SummaryPrion disease is a fatal brain disease caused by misfolding of the prion protein (PrP). Emerging therapies for prion disease seek to reduce the amount of PrP produced in the brain in order to delay onset of disease or slow progression. Mouse studies have shown that if these therapies are initiated too late, their benefit is limited or they may not help at all. Here we measure the half-life of PrP in the mouse brain, and find that it is about 5 days. When drugs are used to lower PrP by cutting up the RNA that encodes PrP, the RNA drops rapidly while the protein lags behind, and does not reach its minimum level until 4 weeks after the drug is dosed. This half-life is about the same regardless of the species of PrP (mouse or human) and whether or not the brain is infected with prions. Cerebrospinal fluid appears to reflect the real-time levels of brain PrP with no appreciable lag. PrP can be measured in colon, which may be useful in animal studies of systemic drugs to lower PrP. PrP turns over more quickly in the presence of a pathogenic genetic variant, the equivalent of the human D178N variant. These findings suggest that clinical trials can monitor PrP in cerebrospinal fluid to look at drug activity, but should plan timepoints far enough post-dose to account for PrPs rate of turnover, and should focus on patients who will survive long enough to benefit from the drug.

neuroscience↗

bioRxiv2022

Diversity-oriented synthesis encoded by deoxyoligonucleotides

Diversity-oriented synthesis (DOS)is a powerful strategy to prepare molecules with underrepresented features in commercial screening collections, resulting in the elucidation of novel biological mechanisms. In parallel to the development of DOS, DNA-encoded libraries (DELs) have emerged as an effective, efficient screening strategy to identify protein binders. Despite recent advancements in this field, most DEL syntheses are limited by the presence of sensitive DNA-based constructs. Here, we describe the design, synthesis, and validation experiments performed for a 3.7 million-member DEL, generated using diverse skeleton architectures with varying exit vectors, derived from DOS, to achieve structural diversity beyond what is possible by varying appendages alone. We will make this DEL available to the academic scientific community to increase access to novel structural features and accelerate early-phase drug discovery.

biochemistry↗

bioRxiv2020

Multimodal small-molecule screening for human prion protein binders

Prion disease is a rapidly progressive neurodegenerative disorder caused by misfolding and aggregation of the prion protein (PrP), and there are currently no therapeutic options. PrP ligands could theoretically antagonize prion formation by protecting the native protein from misfolding or by targeting it for degradation, but no validated small-molecule binders have been discovered to date. We deployed a variety of screening methods in an effort to discover binders of PrP, including 19F-observed and saturation transfer difference (STD) nuclear magnetic resonance (NMR) spectroscopy, differential scanning fluorimetry (DSF), DNA-encoded library selection, and in silico screening. A single benzimidazole compound was confirmed in concentration-response, but affinity was very weak (Kd > 1 mM), and it could not be advanced further. The exceptionally low hit rate observed here suggests that PrP is a difficult target for small-molecule binders. While orthogonal binder discovery methods could yield high affinity compounds, non-small-molecule modalities may offer independent paths forward against prion disease.

biochemistry↗

bioRxiv2019

Characterization of the prion protein binding properties of antisense oligonucleotides

Antisense oligonucleotides (ASOs) designed to lower prion protein (PrP) expression in the brain through RNAse H1-mediated degradation of PrP RNA are in development as prion disease therapeutics. ASOs were previously reported to sequence-independently interact with PrP and inhibit prion accumulation in cell culture, yet in vivo studies using a new generation of ASOs found that only PrP-lowering sequences were effective at extending survival. Cerebrospinal fluid (CSF) PrP has been proposed as a pharmacodynamic biomarker for trials of such ASOs, but is only interpretable if PrP lowering is indeed the relevant mechanism of action in vivo and if measurement of PrP is unconfounded by any PrP-ASO interaction. Here we examine the PrP-binding and antiprion properties of ASOs in vitro and in cell culture. Binding parameters determined by isothermal titration calorimetry were similar across all ASOs tested, indicating that ASOs of various chemistries bind full-length recombinant PrP with low- to mid-nanomolar affinity in a sequence-independent manner. Nuclear magnetic resonance, dynamic light scattering, and visual inspection of ASO-PrP mixtures suggested, however, that this interaction is characterized by the formation of large aggregates, a conclusion further supported by the salt dependence of the affinity measured by isothermal titration calorimetry. Sequence-independent inhibition of prion accumulation in cell culture was observed. The inefficacy of non-PrP-lowering ASOs against prion disease in vivo may be because their apparent activity in vitro is an artifact of aggregation, or because the concentration of ASOs in relevant compartments within the central nervous system (CNS) quickly drops below the effective concentration for sequence-independent antiprion activity after bolus dosing into CSF. ELISA-based measurements of PrP concentration in human CSF were not impacted by the addition of ASO. These findings support the further development of PrP-lowering ASOs and of CSF PrP as a pharmacodynamic biomarker.

biochemistry↗

bioRxiv2019

Domain-specific quantification of prion protein in cerebrospinal fluid by targeted mass spectrometry

Therapies currently in preclinical development for prion disease seek to lower prion protein (PrP) expression in the brain. Trials of such therapies are likely to rely on quantification of PrP in cerebrospinal fluid (CSF) as a pharmacodynamic biomarker and possibly as a trial endpoint. Studies using PrP ELISA kits have reproducibly shown that CSF PrP is lowered in the symptomatic phase of disease, a potential confounder for reading out the effect of PrP-lowering drugs in symptomatic patients. To date it has been unclear whether the reduced abundance of PrP in CSF results from its incorporation into plaques, retention in intracellular compartments, downregulation as a function of the disease process, or other factors. Because misfolding or proteolytic cleavage could potentially render PrP invisible to ELISA even if its concentration were constant or increasing in disease, we sought to establish an orthogonal method for CSF PrP quantification. We developed a targeted mass spectrometry method based on multiple reaction monitoring (MRM) of nine PrP tryptic peptides quantified relative to known concentrations of isotopically labeled standards. Analytical validation experiments showed process replicate coefficients of variation below 15%, good dilution linearity and recovery, and suitable performance for both CSF and brain homogenate and across humans as well as preclinical species of interest. In N=55 CSF samples from individuals referred to prion surveillance centers with rapidly progressive dementia, all six human PrP peptides, spanning the N- and C-terminal domains of PrP, were uniformly reduced in prion disease cases compared to individuals with non-prion diagnoses. This confirms the findings from ELISA studies, demonstrating that lowered CSF PrP concentration in prion disease is a genuine result of the disease process and not merely an artifact of ELISA-based measurement. We provide a targeted mass spectrometry-based method suitable for preclinical and clinical quantification of CSF PrP as a tool for drug development.

neuroscience↗