Drinkable in situ-forming tough hydrogels for gastrointestinal therapeutics.

Pills are a cornerstone of medicine but can be challenging to swallow. While liquid formulations are easier to ingest, they lack the capacity to localize therapeutics with excipients nor act as controlled release devices. Here we describe drug formulations based on liquid in situ-forming tough (LIFT) hydrogels that bridge the advantages of solid and liquid dosage forms. LIFT hydrogels form directly in the stomach through sequential ingestion of a crosslinker solution of calcium and dithiol crosslinkers, followed by a drug-containing polymer solution of alginate and four-arm poly(ethylene glycol)-maleimide. We show that LIFT hydrogels robustly form in the stomachs of live rats and pigs, and are mechanically tough, biocompatible and safely cleared after 24 h. LIFT hydrogels deliver a total drug dose comparable to unencapsulated drug in a controlled manner, and protect encapsulated therapeutic enzymes and bacteria from gastric acid-mediated deactivation. Overall, LIFT hydrogels may expand access to advanced therapeutics for patients with difficulty swallowing.

Screening in serum-derived medium reveals differential response to compounds targeting metabolism.

A challenge for screening new anticancer drugs is that efficacy in cell culture models is not always predictive of efficacy in patients. One limitation of standard cell culture is a reliance on non-physiological nutrient levels, which can influence cell metabolism and drug sensitivity. A general assessment of how physiological nutrients affect cancer cell response to small molecule therapies is lacking. To address this, we developed a serum-derived culture medium that supports the proliferation of diverse cancer cell lines and is amenable to high-throughput screening. We screened several small molecule libraries and found that compounds targeting metabolic enzymes were differentially effective in standard compared to serum-derived medium. We exploited the differences in nutrient levels between each medium to understand why medium conditions affected the response of cells to some compounds, illustrating how this approach can be used to screen potential therapeutics and understand how their efficacy is modified by available nutrients.

Screening in serum-derived medium reveals differential response to compounds targeting metabolism.

A challenge for screening new candidate drugs to treat cancer is that efficacy in cell culture models is not always predictive of efficacy in patients. One limitation of standard cell culture is a reliance on non-physiological nutrient levels to propagate cells. Which nutrients are available can influence how cancer cells use metabolism to proliferate and impact sensitivity to some drugs, but a general assessment of how physiological nutrients affect cancer cell response to small molecule therapies is lacking. To enable screening of compounds to determine how the nutrient environment impacts drug efficacy, we developed a serum-derived culture medium that supports the proliferation of diverse cancer cell lines and is amenable to high-throughput screening. We used this system to screen several small molecule libraries and found that compounds targeting metabolic enzymes were enriched as having differential efficacy in standard compared to serum-derived medium. We exploited the differences in nutrient levels between each medium to understand why medium conditions affected the response of cells to some compounds, illustrating how this approach can be used to screen potential therapeutics and understand how their efficacy is modified by available nutrients.

Compressed phenotypic screens for complex multicellular models and high-content assays.

High-throughput phenotypic screens leveraging biochemical perturbations, high-content readouts, and complex multicellular models could advance therapeutic discovery yet remain constrained by limitations of scale. To address this, we establish a method for compressing screens by pooling perturbations followed by computational deconvolution. Conducting controlled benchmarks with a highly bioactive small molecule library and a high-content imaging readout, we demonstrate increased efficiency for compressed experimental designs compared to conventional approaches. To prove generalizability, we apply compressed screening to examine transcriptional responses of patient-derived pancreatic cancer organoids to a library of tumor-microenvironment (TME)-nominated recombinant protein ligands. Using single-cell RNA-seq as a readout, we uncover reproducible phenotypic shifts induced by ligands that correlate with clinical features in larger datasets and are distinct from reference signatures available in public databases. In sum, our approach enables phenotypic screens that interrogate complex multicellular models with rich phenotypic readouts to advance translatable drug discovery as well as basic biology.

Transcriptional Profiling Supports the Notochordal Origin of Chordoma and Its Dependence on a TGFB1-TBXT Network.

Chordoma is a rare malignant tumor demonstrating notochordal differentiation. It is dependent on brachyury (TBXT), a hallmark notochordal gene and transcription factor, and shares histologic features and the same anatomic location as the notochord. This study involved a molecular comparison of chordoma and notochord to identify dysregulated cellular pathways. The lack of a molecular reference from appropriate control tissue limits our understanding of chordoma and its relationship to notochord. Therefore, an unbiased comparison of chordoma, human notochord, and an atlas of normal and cancerous tissue was conducted using gene expression profiling to clarify the chordoma/notochord relationship and potentially identify novel drug targets. The study found striking consistency in gene expression profiles between chordoma and notochord, supporting the hypothesis that chordoma develops from notochordal remnants. A 12-gene diagnostic chordoma signature was identified and the TBXT/transforming growth factor beta (TGF-ß)/SOX6/SOX9 pathway was hyperactivated in the tumor, suggesting that pathways associated with chondrogenesis were a central driver of chordoma development. Experimental validation in chordoma cells confirmed these findings and emphasized the dependence of chordoma proliferation and survival on TGF-ß. The computational and experimental evidence provided the first molecular connection between notochord and chordoma and identified core members of a chordoma regulatory pathway involving TBXT. This pathway provides new therapeutic targets for this unique malignant neoplasm and highlights TGF-ß as a prime druggable candidate.

Massively parallel pooled screening reveals genomic determinants of nanoparticle delivery.

To accelerate the translation of cancer nanomedicine, we used an integrated genomic approach to improve our understanding of the cellular processes that govern nanoparticle trafficking. We developed a massively parallel screen that leverages barcoded, pooled cancer cell lines annotated with multiomic data to investigate cell association patterns across a nanoparticle library spanning a range of formulations with clinical potential. We identified both materials properties and cell-intrinsic features that mediate nanoparticle-cell association. Using machine learning algorithms, we constructed genomic nanoparticle trafficking networks and identified nanoparticle-specific biomarkers. We validated one such biomarker: gene expression of SLC46A3, which inversely predicts lipid-based nanoparticle uptake in vitro and in vivo. Our work establishes the power of integrated screens for nanoparticle delivery and enables the identification and utilization of biomarkers to rationally design nanoformulations.

Deadenylase-dependent mRNA decay of GDF15 and FGF21 orchestrates food intake and energy expenditure.

Hepatokines, secretory proteins from the liver, mediate inter-organ communication to maintain a metabolic balance between food intake and energy expenditure. However, molecular mechanisms by which hepatokine levels are rapidly adjusted following stimuli are largely unknown. Here, we unravel how CNOT6L deadenylase switches off hepatokine expression after responding to stimuli (e.g., exercise and food) to orchestrate energy intake and expenditure. Mechanistically, CNOT6L inhibition stabilizes hepatic Gdf15 and Fgf21 mRNAs, increasing corresponding serum protein levels. The resulting upregulation of GDF15 stimulates the hindbrain to suppress appetite, while increased FGF21 affects the liver and adipose tissues to induce energy expenditure and lipid consumption. Despite the potential of hepatokines to treat metabolic disorders, their administration therapies have been challenging. Using small-molecule screening, we identified a CNOT6L inhibitor enhancing GDF15 and FGF21 hepatokine levels, which dramatically improves diet-induced metabolic syndrome. Our discovery, therefore, lays the foundation for an unprecedented strategy to treat metabolic syndrome.

Computationally guided high-throughput design of self-assembling drug nanoparticles.

Nanoformulations of therapeutic drugs are transforming our ability to effectively deliver and treat a myriad of conditions. Often, however, they are complex to produce and exhibit low drug loading, except for nanoparticles formed via co-assembly of drugs and small molecular dyes, which display drug-loading capacities of up to 95%. There is currently no understanding of which of the millions of small-molecule combinations can result in the formation of these nanoparticles. Here we report the integration of machine learning with high-throughput experimentation to enable the rapid and large-scale identification of such nanoformulations. We identified 100 self-assembling drug nanoparticles from 2.1 million pairings, each including one of 788 candidate drugs and one of 2,686 approved excipients. We further characterized two nanoparticles, sorafenib-glycyrrhizin and terbinafine-taurocholic acid both ex vivo and in vivo. We anticipate that our platform can accelerate the development of safer and more efficacious nanoformulations with high drug-loading capacities for a wide range of therapeutics.

High-throughput phenotypic screen and transcriptional analysis identify new compounds and targets for macrophage reprogramming.

Macrophages are plastic and, in response to different local stimuli, can polarize toward multi-dimensional spectrum of phenotypes, including the pro-inflammatory M1-like and the anti-inflammatory M2-like states. Using a high-throughput phenotypic screen in a library of ~4000 FDA-approved drugs, bioactive compounds and natural products, we find ~300 compounds that potently activate primary human macrophages toward either M1-like or M2-like state, of which ~30 are capable of reprogramming M1-like macrophages toward M2-like state and another ~20 for the reverse repolarization. Transcriptional analyses of macrophages treated with 34 non-redundant compounds identify both shared and unique targets and pathways through which the tested compounds modulate macrophage activation. One M1-activating compound, thiostrepton, is able to reprogram tumor-associated macrophages toward M1-like state in mice, and exhibit potent anti-tumor activity. Our compound-screening results thus help to provide a valuable resource not only for studying the macrophage biology but also for developing therapeutics through modulating macrophage activation.

Synthesis of Potent Cytotoxic Epidithiodiketopiperazines Designed for Derivatization.

We describe our design, synthesis, and chemical study of a set of functional epidithiodiketopiperazines (ETPs) and evaluation of their activity against five human cancer cell lines. Our structure-activity relationship-guided substitution of ETP alkaloids offers versatile derivatization while maintaining potent anticancer activity, offering exciting opportunity for their use as there are no examples of complex and potently anticancer (nM) ETPs being directly used as conjugatable probes or warheads. Our synthetic solutions to strategically designed ETPs with functional linkers required advances in stereoselective late-stage oxidation and thiolation chemistry in complex settings, including the application of novel reagents for dihydroxylation and cis-sulfidation of diketopiperazines. We demonstrate that complex ETPs equipped with a strategically substituted azide functional group are readily derivatized to the corresponding ETP-triazoles without compromising anticancer activity. Our chemical stability studies of ETPs along with cytotoxic evaluation of our designed ETPs against A549, DU 145, HeLa, HCT 116, and MCF7 human cancer cell lines provide insights into the impact of structural features on potency and chemical stability, informing future utility of ETPs in chemical and biological studies.

Identification and local delivery of vasodilators for the reduction of ureteral contractions.

Kidney stones and ureteral stents can cause ureteral colic and pain. By decreasing contractions in the ureter, clinically prescribed oral vasodilators may improve spontaneous stone passage rates and reduce the pain caused by ureteral stenting. We hypothesized that ureteral relaxation can be improved via the local administration of vasodilators and other smooth muscle relaxants. Here, by examining 18 candidate small molecules in an automated screening assay to determine the extent of ureteral relaxation, we show that the calcium channel blocker nifedipine and the Rho-kinase inhibitor ROCKi significantly relax human ureteral smooth muscle cells. We also show, by using ex vivo porcine ureter segments and sedated pigs that, with respect to the administration of a placebo, the local delivery of a clinically deployable formulation of the two drugs reduced ureteral contraction amplitude and frequency by 90% and 50%, respectively. Finally, we show that standard oral vasodilator therapy reduced contraction amplitude by only 50% and had a minimal effect on contraction frequency. Locally delivered ureteral relaxants therefore may improve ureter-related conditions.

Small-Molecule and CRISPR Screening Converge to Reveal Receptor Tyrosine Kinase Dependencies in Pediatric Rhabdoid Tumors.

Cancer is often seen as a disease of mutations and chromosomal abnormalities. However, some cancers, including pediatric rhabdoid tumors (RTs), lack recurrent alterations targetable by current drugs and need alternative, informed therapeutic options. To nominate potential targets, we performed a high-throughput small-molecule screen complemented by a genome-scale CRISPR-Cas9 gene-knockout screen in a large number of RT and control cell lines. These approaches converged to reveal several receptor tyrosine kinases (RTKs) as therapeutic targets, with RTK inhibition effective in suppressing RT cell growth in vitro and against a xenograft model in vivo. RT cell lines highly express and activate (phosphorylate) different RTKs, creating dependency without mutation or amplification. Downstream of RTK signaling, we identified PTPN11, encoding the pro-growth signaling protein SHP2, as a shared dependency across all RT cell lines. This study demonstrates that large-scale perturbational screening can uncover vulnerabilities in cancers with "quiet" genomes.

Total Synthesis and Anti-Cancer Activity of All Known Communesin Alkaloids and Related Derivatives.

A unified enantioselective total synthesis and anticancer evaluation of all known epoxide-containing communesin alkaloids and related derivatives is described. Our synthesis is predicated on the convergent and modular diazene-directed assembly of two complex fragments to secure the critical C3a-C3a' linkage followed by a guided biomimetic aminal reorganization to deliver the heptacyclic core of these alkaloids. Concise enantioselective syntheses of the fragments were devised, with highlights including the application of a rationally designed sulfinamide chiral auxiliary, an efficient calcium trifluoromethanesulfonate promoted intramolecular amination, and a diastereoselective epoxidation that simultaneously converts the new chiral auxiliary to a versatile amine protective group. The modularity of our convergent approach enabled the rapid synthesis of all epoxide-containing members of the communesin family from a single heterodimeric intermediate, including the first total synthesis of communesins C-E, and G-I, and facilitated our stereochemical revision of (-)-communesin I, the most recently isolated communesin alkaloid. Furthermore, the generality of our biogenetically inspired heterodimer rearrangement was demonstrated in a guided synthesis of a communesin derivative with an unnatural topology. Finally, we report the first comparative analysis of the anticancer activities of all naturally occurring communesin alkaloids A-I and eight complex derivatives against five human cancer cell lines. From these data, we have identified (-)-communesin B as the most potent natural communesin and discovered that derivatives with N8'-sulfonamide substitution exhibit up to a 10-fold increase in potency over the natural alkaloids.

A High-Throughput Assay for Collagen Secretion Suggests an Unanticipated Role for Hsp90 in Collagen Production.

Collagen overproduction is a feature of fibrosis and cancer, while insufficient deposition of functional collagen molecules and/or the secretion of malformed collagen is common in genetic disorders like osteogenesis imperfecta. Collagen secretion is an appealing therapeutic target in these and other diseases, as secretion directly connects intracellular biosynthesis to collagen deposition and biological function in the extracellular matrix. However, small molecule and biological methods to tune collagen secretion are severely lacking. Their discovery could prove useful not only in the treatment of disease, but also in providing tools for better elucidating mechanisms of collagen biosynthesis. We developed a cell-based, high-throughput luminescent assay of collagen type I secretion and used it to screen for small molecules that selectively enhance or inhibit that process. Among several validated hits, the Hsp90 inhibitor 17-allylaminogeldanamycin (17-AAG) robustly decreases the secretion of collagen-I by our model cell line and by human primary cells. In these systems, 17-AAG and other pan-isoform Hsp90 inhibitors reduce collagen-I secretion post-translationally and are not global inhibitors of protein secretion. Surprisingly, the consequences of Hsp90 inhibitors cannot be attributed to inhibition of the endoplasmic reticulum's Hsp90 isoform, Grp94. Instead, collagen-I secretion likely depends on the activity of cytosolic Hsp90 chaperones, even though such chaperones cannot directly engage nascent collagen molecules. Our results highlight the value of a cell-based high-throughput screen for selective modulators of collagen secretion and suggest an unanticipated role for cytosolic Hsp90 in collagen secretion.

Integrated genetic and pharmacologic interrogation of rare cancers.

Identifying therapeutic targets in rare cancers remains challenging due to the paucity of established models to perform preclinical studies. As a proof-of-concept, we developed a patient-derived cancer cell line, CLF-PED-015-T, from a paediatric patient with a rare undifferentiated sarcoma. Here, we confirm that this cell line recapitulates the histology and harbours the majority of the somatic genetic alterations found in a metastatic lesion isolated at first relapse. We then perform pooled CRISPR-Cas9 and RNAi loss-of-function screens and a small-molecule screen focused on druggable cancer targets. Integrating these three complementary and orthogonal methods, we identify CDK4 and XPO1 as potential therapeutic targets in this cancer, which has no known alterations in these genes. These observations establish an approach that integrates new patient-derived models, functional genomics and chemical screens to facilitate the discovery of targets in rare cancers.

A genetic basis for the variation in the vulnerability of cancer to DNA damage.

Radiotherapy is not currently informed by the genetic composition of an individual patient's tumour. To identify genetic features regulating survival after DNA damage, here we conduct large-scale profiling of cellular survival after exposure to radiation in a diverse collection of 533 genetically annotated human tumour cell lines. We show that sensitivity to radiation is characterized by significant variation across and within lineages. We combine results from our platform with genomic features to identify parameters that predict radiation sensitivity. We identify somatic copy number alterations, gene mutations and the basal expression of individual genes and gene sets that correlate with the radiation survival, revealing new insights into the genetic basis of tumour cellular response to DNA damage. These results demonstrate the diversity of tumour cellular response to ionizing radiation and establish multiple lines of evidence that new genetic features regulating cellular response after DNA damage can be identified.

Inhibitors of Glycogen Synthase Kinase 3 with Exquisite Kinome-Wide Selectivity and Their Functional Effects.

The mood stabilizer lithium, the first-line treatment for bipolar disorder, is hypothesized to exert its effects through direct inhibition of glycogen synthase kinase 3 (GSK3) and indirectly by increasing GSK3's inhibitory serine phosphorylation. GSK3 comprises two highly similar paralogs, GSK3α and GSK3ß, which are key regulatory kinases in the canonical Wnt pathway. GSK3 stands as a nodal target within this pathway and is an attractive therapeutic target for multiple indications. Despite being an active field of research for the past 20 years, many GSK3 inhibitors demonstrate either poor to moderate selectivity versus the broader human kinome or physicochemical properties unsuitable for use in in vitro systems or in vivo models. A nonconventional analysis of data from a GSK3ß inhibitor high-throughput screening campaign, which excluded known GSK3 inhibitor chemotypes, led to the discovery of a novel pyrazolo-tetrahydroquinolinone scaffold with unparalleled kinome-wide selectivity for the GSK3 kinases. Taking advantage of an uncommon tridentate interaction with the hinge region of GSK3, we developed highly selective and potent GSK3 inhibitors, BRD1652 and BRD0209, which demonstrated in vivo efficacy in a dopaminergic signaling paradigm modeling mood-related disorders. These new chemical probes open the way for exclusive analyses of the function of GSK3 kinases in multiple signaling pathways involved in many prevalent disorders.

Correlating chemical sensitivity and basal gene expression reveals mechanism of action.

Changes in cellular gene expression in response to small-molecule or genetic perturbations have yielded signatures that can connect unknown mechanisms of action (MoA) to ones previously established. We hypothesized that differential basal gene expression could be correlated with patterns of small-molecule sensitivity across many cell lines to illuminate the actions of compounds whose MoA are unknown. To test this idea, we correlated the sensitivity patterns of 481 compounds with ∼19,000 basal transcript levels across 823 different human cancer cell lines and identified selective outlier transcripts. This process yielded many novel mechanistic insights, including the identification of activation mechanisms, cellular transporters and direct protein targets. We found that ML239, originally identified in a phenotypic screen for selective cytotoxicity in breast cancer stem-like cells, most likely acts through activation of fatty acid desaturase 2 (FADS2). These data and analytical tools are available to the research community through the Cancer Therapeutics Response Portal.

Harnessing Connectivity in a Large-Scale Small-Molecule Sensitivity Dataset.

UNLABELLED: Identifying genetic alterations that prime a cancer cell to respond to a particular therapeutic agent can facilitate the development of precision cancer medicines. Cancer cell-line (CCL) profiling of small-molecule sensitivity has emerged as an unbiased method to assess the relationships between genetic or cellular features of CCLs and small-molecule response. Here, we developed annotated cluster multidimensional enrichment analysis to explore the associations between groups of small molecules and groups of CCLs in a new, quantitative sensitivity dataset. This analysis reveals insights into small-molecule mechanisms of action, and genomic features that associate with CCL response to small-molecule treatment. We are able to recapitulate known relationships between FDA-approved therapies and cancer dependencies and to uncover new relationships, including for KRAS-mutant cancers and neuroblastoma. To enable the cancer community to explore these data, and to generate novel hypotheses, we created an updated version of the Cancer Therapeutic Response Portal (CTRP v2). SIGNIFICANCE: We present the largest CCL sensitivity dataset yet available, and an analysis method integrating information from multiple CCLs and multiple small molecules to identify CCL response predictors robustly. We updated the CTRP to enable the cancer research community to leverage these data and analyses.

Concise Total Synthesis of (+)-Luteoalbusins A and B.

The first total synthesis of (+)-luteoalbusins A and B is described. Highly regio- and diastereoselective chemical transformations in our syntheses include a Friedel-Crafts C3-indole addition to a cyclotryptophan-derived diketopiperazine, a late-stage diketopiperazine dihydroxylation, and a C11-sulfidation sequence, in addition to congener-specific polysulfane synthesis and cyclization to the corresponding epipolythiodiketopiperazine. We also report the cytoxicity of both alkaloids, and closely related derivatives, against A549, HeLa, HCT116, and MCF7 human cancer cell lines.