Abstract P061: <i>De novo</i> design of importin-<i>a</i>-specific NLS sequences for nuclear-targeted therapeutics
Notice bibliographique
Résumé
Abstract Background: Research focused on the application of nuclear localization sequence (NLS)-based therapeutics has been a topic of intense interest to medicine since the core principles governing nuclear transport were awarded the Nobel Prize in 1999. Despite these efforts, efficient nuclear localization has been difficult. A major obstacle is that NLSs have cationic-net charges (abundance of lysines/arginines) required for appropriate interactions with the nuclear transporter importin-a (Impa). Once in the blood stream, cationic charges can cause strong non-specific cellular uptake and are rapidly cleared from the plasma compartment, which prevents sufficient tumor cell uptake and, hence, nuclear localization. Here, describe a de novo computational approach for generating 43 novel NLSs (based on 3 different NLS classes) that contained amino acid substitutions to achieve net-neutral charge states. We also introduce a novel nuclear isolation-quantitative flow cytometric method for determining nuclear localization efficiency. Material and methods An algorithm was created based on complete interaction binding affinity strengths for 20 x 20 pairs of octapeptides consisting of the 20 common amino acids. 9 PDB files were selected as templates and were comprised of viral, RNA processing, and transcription initiation protein NLSs bound to Impa. In silico alanine scanning on all NLS templates generated rankings on amino acid sensitivities for each position in the sequences. Non-sensitive residues were then subjected to residue scanning for generating net-neutral charged NLSs. Computational docking studies generated predictive binding scores relative to wild type NLSs. Favorable NLS candidates were genetically fused to GFP. In contrast to utilizing fluorescence microscopy, which cannot determine nuclear localization efficiency, we created a method to isolate nuclei from transiently transfected CHOK1 cells and quantify nuclear localization by flow cytometry. Results 2-8 net-neutral NLSs with good binding for Imga could be generated for each PDB file. The net-neutral mutants often contained an abundance of glutamic and/or aspartic acid substitutions, and were able to bind to Impa residues to compensate for the replaced amino acids. Transfected GFP-NLS constructs displayed variable fluorescence expression kinetics. Therefore, we created an in-house plasma membrane lysis protocol to isolate intact nuclei. Quantitative evaluations are currently underway by evaluating nuclei fluorescence by flow cytometry at various time points. Thus far, the tested GFP-NLSs are able to localize to the nucleus. Conclusions An important objective of computational protein design is the generation of high affinity peptides as a precursor to the development of therapeutics, and as a tool to aid researchers in understanding governing interaction principles of specific complexes. We have achieved both the development of potential NLS peptides for overcoming the cationic-sequestration barrier, and for understanding novel NLS principles to further advance the NLS-therapeutics field. Citation Format: Olga Bednova, Alexis Rioux-Chevalier, Dipika Patel, Mathieu Boudreau, Jeffrey Victor Leyton. De novo design of importin-a-specific NLS sequences for nuclear-targeted therapeutics [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P061.
Récupéré en direct depuis OpenAlex et désinversé. Les résumés ne sont pas conservés dans cette base de données : les index inversés représentent 8,6 Go des 9,3 Go de texte de la base, et le serveur dispose de 13 Go libres.
Comment cette classification a été obtenuedéplier
Prédiction machine sur la base complète
Imitation des enseignantsNi prévalence calibrée, ni vérité terrain. Validation humaine à venir. Le volet Gemma est une étiquette directe du modèle pour chaque travail de la base, lue sur la notice réduite au titre. Le volet Codex est un classifieur appris des 10 348 étiquettes directes de Codex et calibré sur les taux pondérés de l'échantillon; les champs sans appui suffisant ne portent aucun appel Codex. Le mode candidate est l'union des deux volets; le consensus est leur intersection. Ces sorties portent le statut machine_predicted_unvalidated et ne sont pas des étiquettes humaines.
Scores du classifieur distillé par catégorie (deux têtes)
| Catégorie | Codex | Gemma |
|---|---|---|
| Métarecherche | 0,000 | 0,001 |
| Méta-épidémiologie (sens strict) | 0,000 | 0,000 |
| Méta-épidémiologie (sens large) | 0,000 | 0,001 |
| Bibliométrie | 0,000 | 0,000 |
| Études des sciences et des technologies | 0,000 | 0,000 |
| Communication savante | 0,001 | 0,000 |
| Science ouverte | 0,001 | 0,000 |
| Intégrité de la recherche | 0,001 | 0,001 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,003 | 0,001 |
Scores machine (provisoires)
Les deux têtes enseignantes du modèle étudiant, lues sur ce travail. Un score ordonne la base pour la relecture; il n'affirme jamais une catégorie, et le statut de validation accompagne chaque rangée tel quel.
Scores de référence d'un modèle non mature (critères de maturité non atteints, 7 itérations). Un score ordonne; il n'affirme jamais une catégorie.
score_only:v0-immature-baseline · tel quel depuis la passe de notation : score_only signifie que le nombre peut ordonner les travaux, et qu'aucune étiquette de catégorie n'en découleClassification
machine, non validéePrédiction automatique; un appel candidat d’une seule source (Gemma direct ou Codex distillé), pas un consensus.
Le détail, modèle par modèle et score par score, se trouve en fin de page sous « Comment cette classification a été obtenue ».