Biology subjects

Hussain, R.

Publications and source records attributed to Hussain, R..

At least 19 recordsLinked to original sources

bioRxiv2026

Transient Vascular Cell Integration and Intrinsic Media-Stimulated Endothelial Activation Reveal Diabetic Retinopathy Signatures in Human Retinal Organoids

Retinal organoids (ROs) model human retinogenesis but remain avascular and lack immune components, limiting their utility for studying vascular retinopathies. We generated vascular organoids (VOrs) from human pluripotent stem cells and evaluated their integration into developing ROs. Dissociated VOr cells (day 12 or day 30) were seeded onto 2D retinal neuroepithelium before 3D transition, followed by long-term culture to day 210. VOr derived cells migrated into ROs and transiently persisted. V30 ROs preserved retinal lamination, major neuronal subtypes and light evoked retinal ganglion cell responses comparable to non vascularised controls, whereas media-only ROs cultured in proangiogenic conditions showed structural disorganisation and reduced neuronal survival. Single-cell RNA sequencing revealed an endogenous, inflammatory endothelial-like population with partial endothelial-to-mesenchymal features and a perivascular fibroblast-like cluster in pro-angiogenic media only RO controls. The endothelial-like cell cluster was significantly enriched for genes upregulated in human retinal endothelial cells exposed to high glucose, indicating a diabetic retinopathy like transcriptional state. Microglia like and border associated macrophage populations were present across all conditions, with developmental shifts in morphology and marker expression. Our findings define conditions under which vascular cells can be incorporated into human ROs without disrupting retinal architecture, while uncovering an intrinsic capacity for inflammatory endothelial activation that partially recapitulates diabetic retinopathy. This work advances the engineering of vascularised, immunocompetent retinal models and highlights endogenous vascular immune programmes that must be considered when modelling retinal disease in vitro.

neuroscience↗

bioRxiv2026

Molecular and functional characterization of CYP6AN1 orthologs involved in capsaicin metabolism in two Helicoverpa species

The generalist Helicoverpa armigera and the specialist Helicoverpa assulta are closely related noctuid pests and are among the few insect herbivores capable of feeding on and damaging hot pepper fruits, which contain the defensive compound capsaicin. Cytochrome P450 monooxygenases (P450s) contribute to the metabolism of plant defensive compounds and can facilitate insect adaptation to chemically defended host plants. Here, we identified CYP6AN1 in H. assulta (HassCYP6AN1) and comparatively characterized the CYP6AN1 orthologs from H. armigera (HarmCYP6AN1) and H. assulta. RACE identified one full-length HarmCYP6AN1 transcript and three full-length HassCYP6AN1 transcript isoforms. The HassCYP6AN1 isoforms contained distinct 5' UTRs generated by alternative transcription initiation and splicing but shared an identical coding sequence. Sequence comparisons and phylogenetic analysis supported their assignment as orthologs. Constitutive CYP6AN1 expression was higher in the H. assulta midgut, whereas dietary capsaicin significantly induced CYP6AN1 expression in the H. armigera midgut. Recombinant CYP6AN1 proteins were co-expressed with NADPH-cytochrome P450 reductase in Escherichia coli, and their substrate-metabolizing activities were evaluated using HPLC-based depletion assays. Both orthologs metabolized capsaicin, but HarmCYP6AN1 exhibited an approximately 2.3-fold higher depletion activity than HassCYP6AN1 under the conditions tested. HarmCYP6AN1 also showed P450-content-dependent xanthotoxin depletion, whereas no detectable xanthotoxin metabolism was observed for HassCYP6AN1. These findings establish CYP6AN1 as a component of the capsaicin-metabolizing repertoire of both species and reveal substantial divergence between the orthologs in transcript organization, expression regulation, catalytic activity, and detectable substrate range.

zoology↗

bioRxiv2026

Vessel Spatial Analysis (VeSpA): a tool for whole slide image segmentation, morphometry, and QuPath extension.

Quantifying vascular architecture in histological whole slide images is needed to study tissue organisation, tumour microenvironment biology, and diseaseassociated vascular remodelling. However, vessel analysis in routine immunohistochemistry remains challenging. Available workflows are often manual, require programming expertise, or lack direct integration with digital pathology platforms. We developed VeSpA (Vessel Spatial Analysis), an open-source pipeline and QuPath extension for automated vessel segmentation and morphometric quantification in CD31-stained whole slide images. VeSpA combines configurable signal extraction, using CMYK Yellow channel extraction by default and optional DAB stain deconvolution for H-DAB images, with automatic or percentile-based thresholding, morphological refinement, contour filtering, and lumen filling to generate vessel masks from standard DAB-stained sections. The QuPath extension includes a graphical interface for selecting annotations, TMA cores, or whole images, configuring segmentation parameters, running the Python backend, and importing vessel objects directly into the QuPath hierarchy. For each detected vessel, VeSpA extracts area, major axis length, minor axis length, eccentricity, centroid, and orientation, while also appending summary measurements to parent annotations and TMA cores. Validation against independent pathologist annotations showed that VeSpA achieved segmentation performance close to inter-rater agreement and outperformed yellow channel prompt-based SAM and zero-shot YOLOv8-seg on overlap-based metrics in the tested dataset. VeSpA integrates vessel segmentation, morphometric feature extraction, and QuPath-based visualisation into a single reproducible workflow for vascular quantification in computational pathology and spatial analysis of histological tissue architecture.

pathology↗

bioRxiv2026

Oxidative Stress Susceptibility, Complement Dysregulation, and Metabolic Reprogramming in CFH Y402H Patient-Derived Choriocapillaris Endothelial Cells

Age-related macular degeneration (AMD) is a leading cause of central vision loss. Immunofluorescence and gene expression studies in human donor eyes have shown that choriocapillaris endothelial cells (CECs) are lost before retinal pigment epithelium (RPE) degeneration, leaving extracellular matrix-filled empty lumens known as "ghost vessels." To investigate disease mechanisms, we generated CECs from patient-specific induced pluripotent stem cells (iPSCs) carrying the high-risk CFH Y402H variant and CRISPR-Cas9-corrected isogenic controls. The iPSC-derived CECs expressed canonical endothelial markers, formed fenestrations, maintained barrier function, and assembled capillary-like structures. Although baseline metabolism was preserved, Y402H CECs showed heightened sensitivity to hydroquinone-induced oxidative stress, with increased cytotoxicity and deposition of the complement membrane attack complex (C5b-9). RNA sequencing revealed oxidative stress-driven upregulation of lipid biosynthesis, mTORC signalling, endothelial-to-mesenchymal transition, and angiogenic pathways, alongside an imbalance in the complement pathway. These findings demonstrate that the CFH Y402H polymorphism increases CEC vulnerability to environmental stress, linking complement dysregulation and metabolic reprogramming to choriocapillaris dysfunction in AMD and highlighting CECs as a potential therapeutic target.

Cell Biology↗

bioRxiv2026

Protein disorder controls allostery in DNA

Metabolism, gene expression, and signaling all require the adaptation of protein activity to the mixture of reactants and products in a cell. This trait to adapt, called allostery, is hardwired in the structure of proteins. Binding a ligand at one location in a protein can change distant locations, thus tuning protein activity. How allostery works has been subject of intense research since its discovery sixty years ago. The challenge is to understand the order of events that follow ligand-binding in the three-dimensional architecture of proteins. Here we simplify this task by studying allostery in DNA, a nearly one-dimensional system. DNA can transmit allosteric signals over many nanometers to generate cooperativity in the binding of transcription factors, an archetype of the long-range action of allostery. We found that binding of the transcription factor ComK amplifies intrinsic microsecond structural fluctuations in DNA many nanometers distant from the binding site. Yet, it is not protein binding per se, but the intrinsically disordered region (IDR) of the protein that amplifies these fluctuations. IDR removal does not only rigidify DNA, but it also abolishes allostery. The result is a structurally distorted protein-DNA complex that lost its function. These findings have important implications for our understanding of transcription activation and suggest a new functional role for IDRs in transcription factors.

biophysics↗

bioRxiv2025

Xeno-free human iPSC-derived prostate organoid platform for multilineage differentiation and genetic manipulation

Current prostate organoid models rely on tissue-derived material or animal components and lack epithelial and stromal complexity. We defined a xeno-free system to generate human prostate organoids from induced pluripotent stem cells with consistent multilineage differentiation. Floating organoids self-organize into epithelial and stromal domains with basal, luminal, neuroendocrine, fibroblast, and smooth muscle markers. In an alternative modular co-culture system, engineered epithelial progenitors are aggregated with wild-type mesenchymal progenitors, enabling compartment-specific manipulation. Androgen receptor-overexpressing organoids showed increased epithelial AR and PSA expression and proliferation. Single-cell transcriptomics, together with qPCR and immunostaining, confirmed prostate lineage specification and tissue organization. This new xeno-free platform provides a reproducible, scalable, and genetically tractable model to study in-vitro prostate lineage programs, epithelial-stromal interactions, and disease biology. Graphic AbstractThis study describes the generation of prostate organoids from human iPSCs. iPSCs, including those reprogrammed from patients carrying germline mutations, can be differentiated into prostate organoids either through monoculture or by co-culturing endodermal cells with mesenchymal progenitors. Genetic manipulation can be introduced before endoderm specification to model cancer drivers. The resulting multi-lineage organoids exhibit distinct epithelial (AR, NKX3.1, PSA, CK8/18) and stromal (VIM, -SMA) compartments, providing a versatile platform for developmental studies, disease modelling, drug screening, and biomarker discovery. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/683654v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@1cc13eborg.highwire.dtl.DTLVardef@12fe2a4org.highwire.dtl.DTLVardef@c89d64org.highwire.dtl.DTLVardef@d541e0_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2025

Engineered heat-stable variants of Trypanosoma cruzi flagellar protein Tc24 enable serological detection of Chagas disease

Chagas disease, caused by the protozoan Trypanosoma cruzi, is one of the major neglected diseases globally, killing 12,000 people per year and silently infecting an estimated 7 million people worldwide. Current diagnostic methods are limited by cost, complexity and cold-chain requirements. The T. cruzi flagellar protein Tc24 is a promising antigen for serological tests but suffers of poor solubility and heat stability. Here, we computationally engineered and produced three variants of Tc24, which exhibit remarkable heat stability, up to 69{degrees}C, and express with higher solubility in E. coli compared to the wild-type protein, reducing production costs, eliminating the need for a cold chain and therefore facilitating cost-effective production and storage without refrigeration and even in lyophilised form. These variants remained remarkably stable for 70 days in solution at 25{degrees}C and successfully detected antibodies in human sera samples from Chagas disease patients from Northern and Southern regions of Latin America, demonstrating the preservation of their antigenicity. The best-performing engineered variant was incorporated into a prototype of lateral flow test, demonstrating potential for rapid, affordable and accessible Chagas disease diagnostics in resource-limited settings.

biochemistry↗

bioRxiv2025

Identification of new KCNT1-epilepsy drugs by in silico, cell and Drosophila modelling.

ObjectiveHyperactive KCNT1 potassium channels, caused by gain-of-function mutations, are associated with a range of epilepsy disorders. Patients typically experience drug-resistant seizures and in cases with infantile onset, developmental regression can follow. KCNT1-related disorders include epilepsy of infancy with migrating focal seizures and sleep related hypermotor epilepsy. There are currently no effective treatments for KCNT1-epilepsies, but suppressing over-active channels poses a potential strategy. MethodsUsing KCNT1 channel structural data, we in silico screened a library of known drugs for those predicted to block the channel pore to reduce the current amplitude and inhibit channel activity. ResultsEight known drugs were investigated in vitro for their effects on patient-specific mutant KCNT1 channels, with four drugs showing significant reduction of K+ current amplitudes. The action of the four drugs was then analyzed in vivo and two were found to reduce the seizure phenotype in humanized Drosophila KCNT1-epilepsy models. InterpretationThis study identified two known drugs, antrafenine and nelfinavir mesylate, that reduce KCNT1 channel activity and reduce seizure activity in whole animals, suggesting their potential use as new treatments for KCNT1-epilepsy. The sequential in silico, in vitro and in vivo mechanism-based drug selection strategy used here may have broader application for other human disorders where a disease mechanism has been identified.

pharmacology and toxicology↗

bioRxiv2024

Adapting protein language models for structure-conditioned design

Generative models for protein design trained on experimentally determined structures have proven useful for a variety of design tasks. However, such methods are limited by the quantity and diversity of structures used for training, which represent a small, biased fraction of protein space. Here, we describe proseLM, a method for protein sequence design based on adaptation of protein language models to incorporate structural and functional context. We show that proseLM benefits from the scaling trends of underlying language models, and that the addition of non-protein context - nucleic acids, ligands, and ions - improves recovery of native residues during design by 4-5% across model scales. These improvements are most pronounced for residues that directly interface with non-protein context, which are faithfully recovered at rates >70% by the most capable proseLM models. We experimentally validated proseLM by optimizing the editing efficiency of genome editors in human cells, achieving a 50% increase in base editing activity, and by redesigning therapeutic antibodies, resulting in a PD-1 binder with 2.2 nM affinity.

bioengineering↗

bioRxiv2024

Design of highly functional genome editors by modeling the universe of CRISPR-Cas sequences

Gene editing has the potential to solve fundamental challenges in agriculture, biotechnology, and human health. CRISPR-based gene editors derived from microbes, while powerful, often show significant functional tradeoffs when ported into non-native environments, such as human cells. Artificial intelligence (AI) enabled design provides a powerful alternative with potential to bypass evolutionary constraints and generate editors with optimal properties. Here, using large language models (LLMs) trained on biological diversity at scale, we demonstrate the first successful precision editing of the human genome with a programmable gene editor designed with AI. To achieve this goal, we curated a dataset of over one million CRISPR operons through systematic mining of 26 terabases of assembled genomes and meta-genomes. We demonstrate the capacity of our models by generating 4.8x the number of protein clusters across CRISPR-Cas families found in nature and tailoring single-guide RNA sequences for Cas9-like effector proteins. Several of the generated gene editors show comparable or improved activity and specificity relative to SpCas9, the prototypical gene editing effector, while being 400 mutations away in sequence. Finally, we demonstrate an AI-generated gene editor, denoted as OpenCRISPR-1, exhibits compatibility with base editing. We release OpenCRISPR-1 publicly to facilitate broad, ethical usage across research and commercial applications.

synthetic biology↗

bioRxiv2024

BCG activation of trained immunity is associated with induction of cross reactive COVID-19 antibodies in a BCG vaccinated population.

BackgroundDuring the current COVID-19 pandemic, the rate of morbidity and mortality was considerably lower in BCG vaccinated countries like Pakistan. BCG has been shown to provide cross protection to both disseminated TB as well as non related viral infections in BCG vaccinated children which is consistent with COVID-19 morbidity in the younger age group. Recently, this cross protection was attributed to trained immunity (TI) associated with BCG recall responses in the innate arm of the immune system. Little is known about the longevity of BCG Trained Immunity (TI) beyond early childhood. ObjectiveTo assess the BCG-induced recall responses in healthy individuals by cytokines secreted from the TI network and its potential role in providing cross-protection against COVID-19 and other viral infections. Study DesignIn this cross-sectional study, healthy young adults and adolescents (n=20) were recruited from 16-40 years of age, with no prior history of TB treatment, autoimmune, or chronic inflammatory condition. MethodsBCG-induced cytokine responses were assessed using prototypic markers for cells of the TI network {macrophages [M1 (TNF, IFN{gamma}), M2 (IL10)], NK (IL2), Gamma delta ({gamma}{delta}) T (IL17, IL4)} and SARS CoV2 IgG antibodies against RBD using short-term (12 hrs.) cultures assay. ResultsSignificant differences were observed in the magnitude of recall responses to BCG with macrophage cytokines showing the highest mean levels of TNF (9148 pg/ml) followed by IL10 (488 pg/ml) and IFN{gamma} (355 pg/ml). The ratio of unstimulated vs.BCG-stimulated cytokines was 132 fold higher for TNF, 40 fold for IL10, and 27 fold for IFN{gamma}. Furthermore, SARS-CoV-2 antibodies were also detected in unstimulated plasma which showed cross reactivity with BCG. ConclusionThe presence of cross reactive antibodies to SARS-CoV-2 and the relative ratio of pro-and anti-inflammatory cytokines secreted by activated TI cellular network may play a pivotal role in protection in the early stages of infection as observed during the COVID-19 pandemic in the younger age groups resulting in lower morbidity and mortality.

immunology↗

bioRxiv2023

Deciphering the spatio-temporal transcriptional and chromatin accessibility of human retinal organoid development at the single cell level

Molecular information on the early stages of human retinal development remains scarce due to limitations in obtaining early human eye samples. Pluripotent stem cell-derived retinal organoids provide an unprecedented opportunity for studying early retinogenesis. Using a combination of single cell RNA-Seq and spatial transcriptomics we present for the first-time a single cell spatio-temporal transcriptome of retinal organoid development. Our data demonstrate that retinal organoids recapitulate key events of retinogenesis including optic vesicle/cup formation, formation of a putative ciliary margin zone, emergence of retinal progenitor cells and their orderly differentiation to retinal neurons. Combining the scRNA-with scATAC-Seq data, we were able to reveal cell-type specific transcription factor binding motifs on accessible chromatin at each stage of organoid development and to show that chromatin accessibility is highly correlated to the developing human retina, but with some differences in the temporal emergence and abundance of some of the retinal neurons.

developmental biology↗

bioRxiv2023

Modelling human KCNT1-epilepsy in Drosophila: a seizure phenotype and drug responses

Mutations in the KCNT1 potassium channel cause severe forms of epilepsy which are resistant to current treatments. In vitro studies have shown that KCNT1-epilepsy mutations are gain of function, significantly increasing K+ current amplitudes. To investigate if Drosophila can be used to model human KCNT1 epilepsy, we generated Drosophila melanogaster lines carrying human KCNT1 with the patient mutation G288S, R398Q or R928C. Expression of each mutant channel in GABAergic neurons gave a seizure phenotype which was sensitive to drugs currently used to treat patients with KCNT1-epilepsy. Cannabidiol showed the greatest reduction of the seizure phenotype while some drugs increased the seizure phenotype. Our study shows that Drosophila can be used to model human KCNT1-epilepsy and potentially used as a tool to assess new treatments for KCNT1 epilepsy.

neuroscience↗

bioRxiv2022

BBIBP-CorV (Sinopharm) vaccination- induced immunity is affected by age, gender and prior COVID-19 and activates responses to spike and other antigens

Long-term solutions against SARS-CoV-2 infections require understanding of immune protection induced by different vaccine COVID-19 formulations. We investigated humoral and cellular immunity induced by Sinopharm (BBIBP-CorV) in a region of high SARS-CoV-2 seroprevalence. Levels of IgG antibodies to SARS-CoV-2 spike protein and its receptor-binding domain (RBD) were determined 24-weeks. Cellular immunity was investigated using a commercially available IFN-{gamma} release assay to SARS-CoV-2 spike (Ag1 and 2) and extended genome antigens (Ag3). Increasing IgG seropositivity to Spike protein and RBD was observed post-vaccination. Seropositivity was reduced in those over 50 years and raised in females and those with prior COVID-19. After 20 weeks post-vaccination, only one third of participants had positive T cell responses to SARS-CoV-2 antigens. Prior COVID-19 impacted IFN{gamma} responses, with reactivity enhanced in those infected earlier. The frequency of IFN{gamma} responses was highest to extended genome antigen set. Overall, BBIBP-CorV- induced antibody responses were impacted by age, gender and prior COVID-19. Cellular immunity was present in a limited number of individuals after 20 weeks but was enhanced by prior infection. This suggests the need for booster vaccinations in older individuals. BBIBP-CorV-induced cellular activation is broader than to spike, requiring further study to understand how to monitor vaccine effectiveness.

immunology↗

bioRxiv2022

CanSeer: A Method for Development and Clinical Translation of Personalized Cancer Therapeutics

Computational modeling and analysis of biomolecular network models annotated with cancer patient-specific multi-omics data can enable the development of personalized therapies. Current endeavors aimed at employing in silico models towards personalized cancer therapeutics remain to be fully translated. In this work, we present "CanSeer" a novel multi-stage methodology for developing in silico models towards clinical translation of personalized cancer therapeutics. The proposed methodology integrates state-of-the-art dynamical analysis of biomolecular network models with patient-specific genomic and transcriptomic data to assess the individualized therapeutic responses to targeted drugs and their combinations. CanSeers translational approach employs transcriptomic data (RNA-seq based gene expressions) with genomic profile (CNVs, SMs, and SVs). Specifically, patient-specific cancer driver genes are identified, followed by the selection of druggable and/or clinically actionable targets for therapeutic interventions. To exemplify CanSeer, we have designed three case studies including (i) lung squamous cell carcinoma, (ii) breast invasive carcinoma, and (iii) ovarian serous cystadenocarcinoma. The case study on lung squamous cell carcinoma concluded that restoration of Tp53 activity together with an inhibition of EGFR as an efficacious combinatorial treatment for patients with Tp53 and EGFR cancer driver genes. The findings from the cancer case study helped identify personalized treatments including APR-246, APR-246+palbociclib, APR-246+osimertinib, APR-246+afatinib, APR-246+osimertinib+dinaciclib, and APR-246+afatinib+dinaciclib. The second case study on breast invasive carcinoma revealed CanSeers potential to elucidate drug resistance against targeted drugs and their combinations including KU-55933, afuresertib, ipatasertib, and KU-55933+afuresertib. Lastly, the ovarian cancer case study revealed the combinatorial efficacy of APR-246+carmustine, and APR-246+dinaciclib for treating ovarian serous cystadenocarcinoma. Taken together, CanSeer outlines a novel method for systematic identification of optimal tailored treatments with mechanistic insights into patient-to-patient variability of therapeutic response, drug resistance mechanism, and cytotoxicity profiling towards personalized medicine.

systems biology↗

bioRxiv2021

Conjunctival epithelial cells resist productive SARS-CoV-2 infection

Although tropism of SARS-CoV-2 for respiratory tract epithelial cells is well established, an open question is whether the conjunctival epithelium is also a target for SARS-CoV-2. Conjunctival epithelial cells, which express viral entry receptors ACE2 and TMPRSS2, constitute the largest exposed epithelium of the ocular surface tissue, and may represent a relevant viral entry route. To address this question, we generated an organotypic air-liquid-interface model of conjunctival epithelium, composed of progenitor, basal and superficial epithelial cells and fibroblasts, which could be maintained successfully up to day 75 of differentiation. Using single-cell RNA Seq, with complementary imaging and virological assays, we observed that while all conjunctival cell types were permissive to SARS-CoV-2 genome expression, a productive infection did not ensue. The early innate immune response to SARS-CoV-2 infection in conjunctival cells was characterised by a robust autocrine and paracrine NF-K{beta} activity, without activation of antiviral interferon signalling. Collectively, these data enrich our understanding of SARS-CoV-2 infection at the human ocular surface, with potential implications for the design of preventive strategies and conjunctival transplants.

cell biology↗

bioRxiv2021

Impact of maternal intermittent fasting during pregnancy on cardiovascular, metabolic and renal function in adult rat offspring

Pregnant Muslim women are exempt from fasting during Ramadan; however a majority are reported to fast. The impact of this form of maternal intermittent fasting (IF) on fetal development and offspring health is not well defined. Using a rat model, we have shown previously that maternal IF results in fetal growth restriction accompanied by changes in placental nutrient transport function. The aim of this study was to assess cardiovascular, metabolic and renal function in adult offspring of IF-exposed dams. Food was withheld from Wistar rats from 17:00 to 09:00 daily throughout pregnancy; controls had ad libitum access to food. Birth weight was unaffected; however male IF pups grew more slowly up to 10 weeks of age (P < 0.01) whereas IF females matched their control counterparts. Systolic blood pressure (SBP), glucose tolerance and basal renal function at 14 weeks were not affected by IF exposure. When offered saline solutions (0.9-2.1%) to drink, females showed a greater salt preference than males (P < 0.01); however there were no differences between dietary groups. A separate group of pups was weaned onto a 4% NaCl diet. SBP increased in IF pups sooner, at 7 weeks (P < 0.01), than controls which became hypertensive from 10 weeks. Renal function did not appear to differ; however markers of renal injury were elevated in IF males (P < 0.05). Maternal IF does not affect resting cardiovascular, metabolic and renal function; but when challenged by dietary salt load male IF offspring are more prone to renal injury.

physiology↗

bioRxiv2021

A conformational switch controlling the toxicity of the prion protein

Prion infections cause conformational changes of PrPC and lead to progressive neurological impairment. Here we show that toxic, prion-mimetic ligands induce an intramolecular R208-H140 hydrogen bond ("H-latch") altering the flexibility of the 2-3 and {beta}2-2 loops of PrPC. Expression of a PrP2Cys mutant mimicking the H-latch was constitutively toxic, whereas a PrPR207A mutant unable to form the H-latch conferred resistance to prion infection. High-affinity ligands that prevented H-latch induction repressed prion-related neurodegeneration in organotypic cerebellar cultures. We then selected phage-displayed ligands binding wild-type PrPC, but not PrP2Cys. These binders depopulated H-latched conformers and conferred protection against prion toxicity. Finally, brain-specific expression of an antibody rationally designed to prevent H-latch formation, prolonged the life of prion-infected mice despite unhampered prion propagation, confirming that the H-latch is causally linked to prion neurotoxicity.

molecular biology↗