Polo-like kinase inhibitors increase AAV production by halting cell cycle progression
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ABSTRACT:
PROVIDER: PRJNA1142858 | ENA |
REPOSITORIES: ENA
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Project description:Recombinant Adeno-associated viruses (rAAV) are commonly used in gene therapy for preclinical research and therapeutic applications. Despite its clinical efficacy, the manufacturing of rAAV faces challenges in productivity and quality, leading to limited availability. In this study, we aimed to identify compounds that increase the capacity of cells to produce AAV9 with a high-throughput small-molecule screening strategy. With the Arrayed Targeted Library for AAV Screening platform, we screened a library of 3,300 small molecules and identified several targets, including cell-cycle modulators, G protein-coupled receptor modulators, histone deacetylate inhibitors, Janus kinase inhibitors, and metabolic modulators. Most notably, we identified Polo-like kinase isoform 1 (PLK1) inhibitors as enhancers of AAV production. PLK1 inhibition using HMN-214 increased AAV production, and was largely consistent across HEK293 cell lines, vector payloads and capsid serotypes. Using cell cycle and RNA-sequencing analysis, we show that PLK1 inhibition halts cells in the G2/M phase and blocks their exit from the M to G1 phase. These findings can be used to develop more cost-effective methods to increase AAV yield during production in the manufacturing process.
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Project description:Protein kinases have proven to be a very productive class of therapeutic targets, and over 90 inhibitors are currently in clinical use primarily for the treatment of cancer. Repurposing these inhibitors as antimalarials could provide an accelerated path to drug development. In this study, we identified BI-2536, a known potent human polo-like kinase 1 inhibitor, with low nanomolar antiplasmodial activity. Screening of additional PLK1 inhibitors revealed further antiplasmodial candidates despite the lack of an obvious orthologue of PLKs in Plasmodium. A subset of these inhibitors was profiled for their in vitro killing profile, and commonalities between the killing rate and inhibition of nuclear replication were noted. A kinase panel screen identified PfNEK3 as a shared target of these PLK1 inhibitors; however, phosphoproteome analysis confirmed distinct signaling pathways were disrupted by two structurally distinct inhibitors, suggesting PfNEK3 may not be the sole target. Genomic analysis of BI-2536-resistant parasites revealed mutations in genes associated with the starvation-induced stress response, suggesting BI-2536 may also inhibit an aminoacyl-tRNA synthetase.
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