Ipomoeassin F Binds Sec61α to Inhibit Protein Translocation.

Ipomoeassin F is a potent natural cytotoxin that inhibits growth of many tumor cell lines with single-digit nanomolar potency. However, its biological and pharmacological properties have remained largely unexplored. Building upon our earlier achievements in total synthesis and medicinal chemistry, we used chemical proteomics to identify Sec61α (protein transport protein Sec61 subunit alpha isoform 1), the pore-forming subunit of the Sec61 protein translocon, as a direct binding partner of ipomoeassin F in living cells. The interaction is specific and strong enough to survive lysis conditions, enabling a biotin analogue of ipomoeassin F to pull down Sec61α from live cells, yet it is also reversible, as judged by several experiments including fluorescent streptavidin staining, delayed competition in affinity pulldown, and inhibition of TNF biogenesis after washout. Sec61α forms the central subunit of the ER protein translocation complex, and the binding of ipomoeassin F results in a substantial, yet selective, inhibition of protein translocation in vitro and a broad ranging inhibition of protein secretion in live cells. Lastly, the unique resistance profile demonstrated by specific amino acid single-point mutations in Sec61α provides compelling evidence that Sec61α is the primary molecular target of ipomoeassin F and strongly suggests that the binding of this natural product to Sec61α is distinctive. Therefore, ipomoeassin F represents the first plant-derived, carbohydrate-based member of a novel structural class that offers new opportunities to explore Sec61α function and to further investigate its potential as a therapeutic target for drug discovery.

A Golgi-localized mannosidase (MAN1B1) plays a non-enzymatic gatekeeper role in protein biosynthetic quality control.

Conformation-based disorders are manifested at the level of protein structure, necessitating an accurate understanding of how misfolded proteins are processed by the cellular proteostasis network. Asparagine-linked glycosylation plays important roles for protein quality control within the secretory pathway. The suspected role for the MAN1B1 gene product MAN1B1, also known as ER mannosidase I, is to function within the ER similar to the yeast ortholog Mns1p, which removes a terminal mannose unit to initiate a glycan-based ER-associated degradation (ERAD) signal. However, we recently discovered that MAN1B1 localizes to the Golgi complex in human cells and uncovered its participation in ERAD substrate retention, retrieval to the ER, and subsequent degradation from this organelle. The objective of the current study was to further characterize the contribution of MAN1B1 as part of a Golgi-based quality control network. Multiple lines of experimental evidence support a model in which neither the mannosidase activity nor catalytic domain is essential for the retention or degradation of the misfolded ERAD substrate Null Hong Kong. Instead, a highly conserved, vertebrate-specific non-enzymatic decapeptide sequence in the luminal stem domain plays a significant role in controlling the fate of overexpressed Null Hong Kong. Together, these findings define a new functional paradigm in which Golgi-localized MAN1B1 can play a mannosidase-independent gatekeeper role in the proteostasis network of higher eukaryotes.

High-Throughput Cellular Thermal Shift Assay Using Acoustic Transfer of Protein Lysates.

Cellular thermal shift assay (CETSA) is a valuable method to confirm target engagement within a complex cellular environment, by detecting changes in a protein's thermal stability upon ligand binding. The classical CETSA method measures changes in the thermal stability of endogenous proteins using immunoblotting, which is low-throughput and laborious. Reverse-phase protein arrays (RPPAs) have been demonstrated as a detection modality for CETSA; however, the reported procedure requires manual processing steps that limit throughput and preclude screening applications. We developed a high-throughput CETSA using an acoustic RPPA (HT-CETSA-aRPPA) protocol that is compatible with 96- and 384-well microplates from start-to-finish, using low speed centrifugation to remove thermally destabilized proteins. The utility of HT-CETSA-aRPPA for guiding structure-activity relationship studies was demonstrated for inhibitors of lactate dehydrogenase A. Additionally, a collection of kinase inhibitors was screened to identify compounds that engage MEK1, a clinically relevant kinase target.

Robotic high-throughput biomanufacturing and functional differentiation of human pluripotent stem cells.

Efficient translation of human induced pluripotent stem cells (hiPSCs) requires scalable cell manufacturing strategies for optimal self-renewal and functional differentiation. Traditional manual cell culture is variable and labor intensive, posing challenges for high-throughput applications. Here, we established a robotic platform and automated all essential steps of hiPSC culture and differentiation under chemically defined conditions. This approach allowed rapid and standardized manufacturing of billions of hiPSCs that can be produced in parallel from up to 90 different patient- and disease-specific cell lines. Moreover, we established automated multi-lineage differentiation and generated functional neurons, cardiomyocytes, and hepatocytes. To validate our approach, we compared robotic and manual cell culture operations and performed comprehensive molecular and cellular characterizations (e.g., single-cell transcriptomics, mass cytometry, metabolism, electrophysiology) to benchmark industrial-scale cell culture operations toward building an integrated platform for efficient cell manufacturing for disease modeling, drug screening, and cell therapy.

Robotic High-Throughput Biomanufacturing and Functional Differentiation of Human Pluripotent Stem Cells.

Efficient translation of human induced pluripotent stem cells (hiPSCs) depends on implementing scalable cell manufacturing strategies that ensure optimal self-renewal and functional differentiation. Currently, manual culture of hiPSCs is highly variable and labor-intensive posing significant challenges for high-throughput applications. Here, we established a robotic platform and automated all essential steps of hiPSC culture and differentiation under chemically defined conditions. This streamlined approach allowed rapid and standardized manufacturing of billions of hiPSCs that can be produced in parallel from up to 90 different patient-and disease-specific cell lines. Moreover, we established automated multi-lineage differentiation to generate primary embryonic germ layers and more mature phenotypes such as neurons, cardiomyocytes, and hepatocytes. To validate our approach, we carefully compared robotic and manual cell culture and performed molecular and functional cell characterizations (e.g. bulk culture and single-cell transcriptomics, mass cytometry, metabolism, electrophysiology, Zika virus experiments) in order to benchmark industrial-scale cell culture operations towards building an integrated platform for efficient cell manufacturing for disease modeling, drug screening, and cell therapy. Combining stem cell-based models and non-stop robotic cell culture may become a powerful strategy to increase scientific rigor and productivity, which are particularly important during public health emergencies (e.g. opioid crisis, COVID-19 pandemic).

Detecting Secretory Proteins by Acoustic Droplet Ejection in Multiplexed High-Throughput Applications.

Nearly one-third of the encoded proteome is comprised of secretory proteins that enable communication between cells and organ systems, playing a ubiquitous role in human health and disease. High-throughput detection of secreted proteins would enhance efforts to identify therapies for secretion-related diseases. Using the Z mutant of alpha-1 antitrypsin as a human secretory model, we have developed 1536-well high-throughput screening assays that utilize acoustic droplet ejection to transfer nanoliter volumes of sample for protein quantification. Among them, the acoustic reverse phase protein array (acoustic RPPA) is a multiplexable, low-cost immunodetection technology for native, endogenously secreted proteins from physiologically relevant model systems like stem cells that is compatible with plate-based instrumentation. Parallel assay profiling with the LOPAC1280 chemical library validated performance and orthogonality between a secreted bioluminescent reporter and acoustic RPPA method by consistently identifying secretory modulators with comparable concentration response relationships. Here, we introduce a robust, multiplexed drug discovery platform coupling extracellular protein quantification by acoustic RPPA with intracellular and cytotoxicity analyses from single wells, demonstrating proof-of-principle applications for human induced pluripotent stem cell-derived hepatocytes.

Canvass: A Crowd-Sourced, Natural-Product Screening Library for Exploring Biological Space.

Natural products and their derivatives continue to be wellsprings of nascent therapeutic potential. However, many laboratories have limited resources for biological evaluation, leaving their previously isolated or synthesized compounds largely or completely untested. To address this issue, the Canvass library of natural products was assembled, in collaboration with academic and industry researchers, for quantitative high-throughput screening (qHTS) across a diverse set of cell-based and biochemical assays. Characterization of the library in terms of physicochemical properties, structural diversity, and similarity to compounds in publicly available libraries indicates that the Canvass library contains many structural elements in common with approved drugs. The assay data generated were analyzed using a variety of quality control metrics, and the resultant assay profiles were explored using statistical methods, such as clustering and compound promiscuity analyses. Individual compounds were then sorted by structural class and activity profiles. Differential behavior based on these classifications, as well as noteworthy activities, are outlined herein. One such highlight is the activity of (-)-2(S)-cathafoline, which was found to stabilize calcium levels in the endoplasmic reticulum. The workflow described here illustrates a pilot effort to broadly survey the biological potential of natural products by utilizing the power of automation and high-throughput screening.

Analysis of serpin secretion, misfolding, and surveillance in the endoplasmic reticulum.

Biological checkpoints are known to function in the cellular nucleus to monitor the integrity of inherited genetic information. It is now understood that posttranslational checkpoint systems operate in numerous biosynthetic compartments where they orchestrate the surveillance of encoded protein structures. This is particularly true for the serpins where opposing, but complementary, systems operate in the early secretory pathway to initially facilitate protein folding and then selectively target the misfolded proteins for proteolytic elimination. A current challenge is to elucidate how this posttranslational checkpoint can modify the severity of numerous loss-of-function and gain-of-toxic-function diseases, some of which are caused by mutant serpins. This chapter provides a description of the experimental methodology by which the fate of a newly synthesized serpin is monitored, and how the processing of asparagine-linked oligosaccharides helps to facilitate both the protein folding and disposal events.

The natural products parthenolide and andrographolide exhibit anti-cancer stem cell activity in multiple myeloma.

Multiple myeloma (MM) is an incurable plasma cell malignancy where nearly all patients succumb to a relapse. The current preclinical models of MM target the plasma cells, constituting the bulk of the tumor, leaving the cancer stem cells to trigger a relapse. Utilizing a three-dimensional tissue culture system where cells were grown in extracellular matrix designed to reconstruct human bone marrow, we tested the anti-multiple myeloma cancer stem cell (MM-CSC) potential of two natural product inhibitors of nuclear factor κB (NFκB). Here we show that parthenolide and andrographolide are potent anti-MM-CSC agents. Both natural products demonstrated preferential toxicity toward MM-CSCs over non-tumorigenic MM cells. Addition of the bone marrow stromal compartment abrogated andrographolide activity while having no effect on parthenolide cytoxicity. This is the first report of a natural product with anti-CSC activity in myeloma, suggesting that it has the potential to improve the survival of patients with MM by eliminating the relapse-causing MM-CSCs.