Psymberin, a marine-derived natural product, induces cancer cell growth arrest and protein translation inhibition

Colorectal cancer (CRC) is the third most prevalent form of cancer in the United States and results in over 50,000 deaths per year. Treatments for metastatic CRC are limited, and therefore there is an unmet clinical need for more effective therapies. In our prior work, we coupled high-throughput chemical screens with patient-derived models of cancer to identify new potential therapeutic targets for CRC. However, this pipeline is limited by (1) the use of cell lines that do not appropriately recapitulate the tumor microenvironment, and (2) the use of patient-derived xenografts (PDXs), which are time-consuming and costly for validation of drug efficacy. To overcome these limitations, we have turned to patient-derived organoids. Organoids are increasingly being accepted as a “standard” preclinical model that recapitulates tumor microenvironment cross-talk in a rapid, cost-effective platform. In the present work, we employed a library of natural products, intermediates, and drug-like compounds for which full synthesis has been demonstrated. Using this compound library, we performed a high-throughput screen on multiple low-passage cancer cell lines to identify potential treatments. The top candidate, psymberin, was further validated, with a focus on CRC cell lines and organoids. Mechanistic and genomics analyses pinpointed protein translation inhibition as a mechanism of action of psymberin. These findings suggest the potential of psymberin as a novel therapy for the treatment of CRC

Assembly of Protein Building Blocks Using a Short Synthetic Peptide

Combining proteins or their defined domains offers new enhanced functions. Conventionally, two proteins are either fused into a single polypeptide chain by recombinant means or chemically cross-linked. However, these strategies can have drawbacks such as poor expression (recombinant fusions) or aggregation and inactivation (chemical cross-linking), especially in the case of large multifunctional proteins. We developed a new linking method which allows site-oriented, noncovalent, yet irreversible stapling of modified proteins at neutral pH and ambient temperature. This method is based on two distinct polypeptide linkers which self-assemble in the presence of a specific peptide staple allowing on-demand and irreversible combination of protein domains. Here we show that linkers can either be expressed or be chemically conjugated to proteins of interest, depending on the source of the proteins. We also show that the peptide staple can be shortened to 24 amino acids still permitting an irreversible combination of functional proteins. The versatility of this modular technique is demonstrated by stapling a variety of proteins either in solution or to surfaces

Figure S2 from Effects and Eradication of <i>Mycoplasma</i> Contamination on Patient-derived Colorectal Cancer Organoid Cultures

IC50 curves for one round of standard of care drug treatment</p

Table S1 from Effects and Eradication of <i>Mycoplasma</i> Contamination on Patient-derived Colorectal Cancer Organoid Cultures

Table of average percent killing for all drugs in high throughput screens</p

Figure S1 from Effects and Eradication of <i>Mycoplasma</i> Contamination on Patient-derived Colorectal Cancer Organoid Cultures

Authentication of organoid lines after passaging through mice</p

FIGURE 2 from Effects and Eradication of <i>Mycoplasma</i> Contamination on Patient-derived Colorectal Cancer Organoid Cultures

Mycoplasma can be successfully cleared from PDO lines by in vivo passaging through immunodeficient mice. A,Mycoplasma test for organoid lines before (+) and after (−) in vivo passaging. Representative positive and negative control PCR products are included within lanes 1 and 2 of the gel on the left. The top band may not appear if the original sample contained a high amount of Mycoplasma. B, Growth comparison of Mycoplasma-positive and -negative lines over 12 days. Images on top compare PDO growth between positive and negative lines on days 0 and 12. Graphs on the bottom show CTG fluorescence of the PDO taken every other day for 12 days.</p

TABLE 1 from Effects and Eradication of <i>Mycoplasma</i> Contamination on Patient-derived Colorectal Cancer Organoid Cultures

Protocol for eliminating Mycoplasma contamination from PDO lines</p

FIGURE 3 from Effects and Eradication of <i>Mycoplasma</i> Contamination on Patient-derived Colorectal Cancer Organoid Cultures

Mycoplasma contamination can change organoid drug sensitivity. A, Dose–response curves for four sets of Mycoplasm-positive and -negative lines for three drugs: oxaliplatin, SN38, and 5-FU. *, P B, High-throughput screens for three sets of Mycoplasm-positive and -negative organoid lines. Percent killing for each drug is indicated by color, with red being the highest percent killing and blue being the lowest. C, Difference in percent killing in high-throughput screen between Mycoplasm-negative and -positive organoid lines.</p

FIGURE 1 from Effects and Eradication of <i>Mycoplasma</i> Contamination on Patient-derived Colorectal Cancer Organoid Cultures

Plasmocin cannot reliably clear Mycoplasma from PDO lines. A,Mycoplasma testing of Mycoplasma-positive lines treated with Plasmocin for 2 weeks according to the manufacturer's protocol. The top bands are a negative control for the PCR. The bottom bands are indicative of the presence of Mycoplasma (lanes 1 and 2). The top band may not appear if the original sample contained a high amount of Mycoplasma. All four lines were tested in triplicate. B, Growth rate of PDO4 after Plasmocin treatment over 14 days. Graphs on the left show CTG fluorescence of the PDO taken over the course of 14 days. Images on right compare PDO growth on days 0 and 14.</p