High-Throughput Screening Identifies Two Novel Small Molecule Enhancers of Recombinant Protein Expression

Abstract

As a primary strategy for production of biological drugs, recombinant proteins produced by transient transfection of mammalian cells are essential for both basic research and industrial production. Here, we established a high-throughput screening platform for improving the expression levels of recombinant proteins. In total, 10,011 small molecule compounds were screened through our platform. After two rounds of screening, we identified two compounds, Apicidin and M-344, that significantly enhanced recombinant protein expression. Both of the selected compounds were histone deacetylase inhibitors, suggesting that the two small molecules increased the expression levels of recombinant proteins by promoting histone acetylation. Moreover, both molecules showed low cytotoxicity. Therefore, our findings suggest that these small molecules may have wide applications in the future.

Increased recombinant AAV production by HEK293 cells using small molecule chemical additives

“…Here we describe a simple and robust method by which viral vector titre, a fundamental critical quality attribute of viral vector production, may be quickly improved in an established and previously optimised suspension culture system. Small molecule enhancers of recombinant protein expression have been extensively used across a wide range of mammalian cell systems to improve transient production performance [8]- [10], [22], [39], [55], [56] . The ease of use and low cost of small molecule enhancers (particularly for chemicals with pronounced biological activity at low dosages) makes them an attractive solution to improving rAAV yield.…”

“…Due to the diverse mechanisms involved in AAV vector production, the panel featured chemicals that exert their influence on recombinant protein expression via a broad range of functional mechanisms. Specifically, we tested chemicals reported to be "chemical chaperones" (TUDCA, TMAO, betaine) [15]- [17] , cell cycle modulators (nocodazole, BI-2536) [18], [19] , caspase inhibitors (z-VAD-fmk) [20] , histone deacetylase inhibitors (NaBu, VPA, M344, apicidin) [10], [21]- [23] , insulin-mimetics (lithium chloride and zinc sulphate) [24] , and proteasome inhibitors (ONX0912, MLN9708, MG132) [25]- [27] .…”

“…In order to evaluate whether specific combinations of effectors could act synergistically to further increase rAAV production, we utilized nocodazole in combination with either z-VAD-fmk or M344 (see Figure 1). M344 is a synthetic analogue of the anti-fungal drug Trichostatin A, an inhibitor of Class I and IIB histone deacetylases, and recently used as an enhancer of recombinant protein expression in mammalian cell culture [10] . Z-VAD-fmk is a pan-caspase inhibitor [20] .…”

“…rAAV is a complex macromolecular product requiring a diverse network of cellular processes and molecular interactions to enable coordinated cellular synthesis -host-cell proteins, transiently expressed viral helper, capsid and replicase genes as well as the single stranded therapeutic viral DNA payload itself [5]- [7] . Small molecule enhancers of recombinant protein production have demonstrated efficacy in a wide variety of mammalian cell lines [3,10,11] and the addition of chemicals as diverse as sodium chloride, sodium butyrate, and soy peptones have been shown in previous studies to improve rAAV production yields [11]- [13] . Targeting of discrete pathways involved in the replication, packaging, and trafficking of viral particles by bioactive small molecule cell culture additives therefore offers a simple and cost-effective way of increasing viral titre and reducing overall production costs.…”

Increased recombinant adeno‐associated virus production by HEK293 cells using small molecule chemical additives

“…rAAV is a complex macromolecular product requiring a diverse network of cellular processes and molecular interactions to enable coordinated cellular synthesis -involving host-cell proteins, transiently expressed viral helper, capsid and replicase genes, as well as the single stranded therapeutic viral DNA payload itself. [5][6][7] Small molecule enhancers of recombinant protein production have demonstrated efficacy in a wide variety of mammalian cell lines [3,8,9] and the addition of chemicals as diverse as sodium chloride, sodium butyrate (NaBu) and soy peptones have been shown in previous studies to improve rAAV production yields. [9][10][11] Targeting of discrete pathways involved in the replication, packaging and trafficking of viral particles by bioactive small molecule cell culture additives offers a simple and cost-effective way of increasing viral titre and reducing overall production costs.…”