Biology subjects

Lin, L. Y.

Publications and source records attributed to Lin, L. Y..

7 recordsLinked to original sources

bioRxiv2024

Exploring the druggability of the UEV domain of human TSG101 in search for broad-spectrum antivirals

The ubiquitin E2 variant domain of TSG101 (TSG101-UEV) plays a pivotal role in protein sorting and virus budding by recognizing PTAP motifs within ubiquitinated proteins. Disruption of TSG101-UEV/PTAP interactions has emerged as a promising strategy for the development of host-oriented broad-spectrum antivirals with low susceptibility to resistance. TSG101 is a challenging target characterized by an extended and flat binding interface, low affinity for PTAP ligands, and complex binding energetics. Here, we assess the druggability of the TSG101-UEV/PTAP binding interface by searching for drug-like inhibitors and evaluating their ability to block PTAP recognition, impair budding, and inhibit viral proliferation. A discovery workflow was established combining in vitro miniaturized HTS assays and a set of cell-based activity assays including high-content bimolecular complementation, virus-like particle release measurement, and antiviral testing in live virus infection. This approach has allowed us to identify a set of chemically diverse molecules that block TSG101-UEV/PTAP binding with IC50s in the low M range, and able to disrupt the interaction between full-length TSG101 and viral proteins in human cells and inhibit viral replication. State-of-the-art molecular docking studies reveal that the active compounds exploit binding hotspots at the PTAP binding site, unlocking the full binding potential of the TSG101-UEV binding pockets. These inhibitors represent promising hits for the development of novel broad-spectrum antivirals through targeted optimization and are also valuable tools for investigating the involvement of ESCRT in the proliferation of different virus families and study the secondary effects induced by the disruption of ESCRT/virus interactions. ImportanceMany viruses rely on the interaction between TSG101 and viral proteins containing PTAP motifs for their proliferation. Here we show that these interactions can be efficiently blocked by drug-like compounds that impair budding and replication of viruses from different families. We have also provided valuable insights into the determinants of high affinity for these small molecule inhibitors that open new avenues for developing the identified candidates into broad-spectrum antivirals with low susceptibility to resistance.

molecular biology↗

bioRxiv2024

Scalable assessment of genome editing off-targets associated with genetic variants

Genome editing with RNA-guided DNA binding factors carries risk of off-target editing at homologous sequences. Genetic variants may introduce sequence changes that increase homology to a genome editing target, thereby increasing risk of off-target editing. Conventional methods to verify candidate off-targets rely on access to cells with genomic DNA carrying these sequences. However, for candidate off-targets associated with genetic variants, appropriate cells for experimental verification may not be available. Here we develop a method, Assessment By Stand-in Off-target LentiViral Ensemble with sequencing (ABSOLVE-seq), to integrate a set of candidate off-target sequences along with unique molecular identifiers (UMIs) in genomes of primary cells followed by clinically relevant gene editor delivery. Gene editing of dozens of candidate off-target sequences may be evaluated in a single experiment with high sensitivity, precision, and power. We provide an open-source pipeline to analyze sequencing data. This approach enables experimental assessment of the influence of human genetic diversity on specificity evaluation during gene editing therapy development.

genomics↗

bioRxiv2023

Gene editing without ex vivo culture evades genotoxicity in human hematopoietic stem cells

Gene editing the BCL11A erythroid enhancer is a validated approach to fetal hemoglobin (HbF) induction for {beta}-hemoglobinopathy therapy, though heterogeneity in edit allele distribution and HbF response may impact its safety and efficacy. Here we compared combined CRISPR-Cas9 endonuclease editing of the BCL11A +58 and +55 enhancers with leading gene modification approaches under clinical investigation. We found that combined targeting of the BCL11A +58 and +55 enhancers with 3xNLS-SpCas9 and two sgRNAs resulted in superior HbF induction, including in engrafting erythroid cells from sickle cell disease (SCD) patient xenografts, attributable to simultaneous disruption of core half E-box/GATA motifs at both enhancers. We corroborated prior observations that double strand breaks (DSBs) could produce unintended on- target outcomes in hematopoietic stem and progenitor cells (HSPCs) such as long deletions and centromere-distal chromosome fragment loss. We show these unintended outcomes are a byproduct of cellular proliferation stimulated by ex vivo culture. Editing HSPCs without cytokine culture bypassed long deletion and micronuclei formation while preserving efficient on-target editing and engraftment function. These results indicate that nuclease editing of quiescent hematopoietic stem cells (HSCs) limits DSB genotoxicity while maintaining therapeutic potency and encourages efforts for in vivo delivery of nucleases to HSCs.

genetics↗

bioRxiv2023

The chromatin factors SET-26 and HCF-1 oppose the histone deacetylase HDA-1 in longevity and gene regulation in C. elegans

SET-26, HCF-1, and HDA-1 are highly conserved chromatin factors with key roles in development and aging. Here we present mechanistic insights into how these factors regulate gene expression and modulate longevity in C. elegans. We show that SET-26 and HCF-1 cooperate to regulate a common set of genes, and both antagonize the histone deacetylase HDA-1 to limit longevity. We propose a model in which SET-26 recruits HCF-1 to chromatin in somatic cells, where they stabilize each other at the promoters of a subset of genes, particularly mitochondrial function genes, and regulate their expression. HDA-1 opposes SET-26 and HCF-1 on the regulation of a subset of their common target genes and in longevity. Our findings suggest that SET-26, HCF-1, and HDA-1 comprise a mechanism to fine-tune gene expression and longevity and likely have important implications for the mechanistic understanding of how these factors function in diverse organisms, particularly in aging biology.

genomics↗

bioRxiv2021

Human genetic diversity modifies therapeutic gene editing off-target potential

CRISPR gene editing holds great promise to modify somatic genomes to ameliorate disease. In silico prediction of homologous sites coupled with biochemical evaluation of possible genomic off-targets may predict genotoxicity risk of individual gene editing reagents. However, standard computational and biochemical methods focus on reference genomes and do not consider the impact of genetic diversity on off-target potential. Here we developed a web application called CRISPRme that explicitly and efficiently integrates human genetic variant datasets with orthogonal genomic annotations to nominate and prioritize off-target sites at scale. The method considers both single-nucleotide variants (SNVs) and indels, accounts for bona fide haplotypes, accepts spacer:protospacer mismatches and bulges, and is suitable for personal genome analyses. We tested the tool with a guide RNA (gRNA) targeting the BCL11A erythroid enhancer that has shown therapeutic promise in clinical trials for sickle cell disease (SCD) and {beta}-thalassemia1. We find that the top candidate off-target site is produced by a non-reference allele common in African-ancestry populations (rs114518452, minor allele frequency (MAF)=4.5%) that introduces a protospacer adjacent motif (PAM) for SpCas9. We validate that SpCas9 generates indels ([~]9.6% frequency) and chr2 pericentric inversions in a strictly allele-specific manner in edited CD34+ hematopoietic stem/progenitor cells (HSPCs), although a high-fidelity Cas9 variant mitigates this off-target. The CRISPRme tool highlights alternative allele-specific off-target editing as a prevalent risk of gRNAs considered for therapeutic gene editing. Our report illustrates how population and private genetic variants should be considered as modifiers of genome editing outcomes. We suggest that variant-aware off-target assessment should be considered in therapeutic genome editing efforts going forward and provide a powerful approach for comprehensive off-target nomination.

genetics↗

bioRxiv2020

Identification and Characterization of a B-Raf Kinase Alpha Helix Critical for the Activity of MEK Kinase in MAPK Signaling

In the MAPK pathway, an oncogenic V600E mutation in B-Raf kinase causes the enzyme to be constitutively active, leading to aberrantly high phosphorylation levels of its downstream effectors, MEK and ERK kinases. The V600E mutation in B-Raf accounts for more than half of all melanomas and [~]3% of all cancers and many drugs target the ATP-binding site of the enzyme for its inhibition. Since B-Raf can develop resistance against these drugs and such drugs can induce paradoxical activation, drugs that target allosteric sites are needed. To identify other potential drug targets, we generated and kinetically characterized an active form of B-RafV600E expressed using a bacterial expression system. In doing so, we identified an alpha helix on B-Raf, found at the B-Raf-MEK interface, that is critical for their interaction and the oncogenic activity of B-RafV600E. We performed binding experiments between B-Raf mutants and MEK using pull downs and biolayer interferometry, and assessed phosphorylation levels of MEK in vitro and in cells as well as its downstream target ERK to show that mutating certain residues on this alpha helix is detrimental to binding and downstream activity. Our results suggest that this B-Raf alpha helix binding site on MEK could be a site to target for drug development to treat B-RafV600E-induced melanomas.

biochemistry↗

bioRxiv2019

Kelch13 and MDR1 Polymorphisms, and Drug Effectiveness at Day 3 after Dihydroartemisinin-Piperaquine Treatment for Plasmodium falciparum Malaria on Bioko Island, Equatorial Guinea: 2014-2017

Artemisinin (ART) combination therapies were introduced on malaria endemic Bioko Island in 2004 through Bioko Island Malaria Control Project. Recently, ART-resistant Plasmodium falciparum strain with Kelch13 (K13) propeller M579I mutation originating from Equatorial Guinea was observed as an increased parasite clearance time on day 3 after dihydroartemisinin-Piperaquine (DHA-PIP) treatment (D3 positivity). Here, we surveyed DHA-PIP effectiveness and molecular markers of drug resistance at D3 after DHA-PIP treatment on Bioko Island from 2014 to 2017. Among the 371 uncomplicated P. falciparum patients, 86.3% (320/471) were successfully followed up at D3. 5.9% (19/320) of patients showed D3 positivity. K13 and MDR1 gene were successfully sequenced from 46 patients collected at D0 (baseline population) and 19 D3-positivity patients. Five non-synonymous K13 mutations (H136N; K189N; K248N; K326E; K332N) were found. There was no statistical difference in the frequency of these K13 mutations between baseline population and D3-positivity samples (p>0.05). Additionally, none of the K13 propeller polymorphisms known to be involved in ART-resistance in Asia or Africa were detected. For MDR1 gene, 38.5% (25/65) carried N86Y mutation; 73.8% (48/65) the Y184F mutation. Parasites surviving DHA-PIP at D3 post-treatment were significantly more likely than the baseline population to carry the N86Y (p <0.05). These results suggest that K13 is not the best predictive molecular marker for ART resistance in Africa. More isolates from cases with delayed parasite clearance after DHA-PIP treatment indicated that in vitro and in vivo monitoring for ART derivatives and ACT partner drugs should be regularly performed on Bioko Island, Equatorial Guinea.

epidemiology↗