Comparison of dose selection based on target engagement versus inhibition of receptor-ligand interaction for checkpoint inhibitors.

Immune checkpoint inhibitors block the interaction between a receptor on one cell and its ligand on another cell, thus preventing the transduction of an immunosuppressive signal. While inhibition of the receptor-ligand interaction is key to the pharmacological activity of these drugs, it can be technically challenging to measure these intercellular interactions directly. Instead, target engagement (or receptor occupancy) is commonly measured, but may not always be an accurate predictor of receptor-ligand inhibition, and can be misleading when used to inform clinical dose projections for this class of drugs. In this study, a mathematical model explicitly representing the intercellular receptor-ligand interaction is used to compare dose prediction based on target engagement or receptor-ligand inhibition for two checkpoint inhibitors, atezolizumab and magrolimab. For atezolizumab, there is little difference between target engagement and receptor-ligand inhibition, but for magrolimab, the model predicts that receptor-ligand inhibition is significantly less than target engagement. The key variables explaining the difference between these two drugs are the relative concentrations of the target receptors and their ligands. Drug-target affinity and receptor-ligand affinity can also have divergent effects on target engagement and inhibition. These results suggest that it is important to consider ligand-receptor inhibition in addition to target engagement and demonstrate the impact of using modeling for efficacious dose estimation.

Insights into the structure-activity relationship of the anticancer compound ZJ-101: A role played by the amide moiety.

ZJ-101, a structurally simplified analog of marine natural product superstolide A, was previously designed and synthesized in our laboratory. In the present study four new analogs of ZJ-101 were designed and synthesized to investigate the structure-activity relationship of the acetamide moiety of the molecule. The biological evaluation showed that the amide moiety is important for the molecule's anticancer activity. Replacing the amide with other functional groups such as a sulfonamide group, a carbamate group, and a urea group resulted in the decrease in anticancer activity.

Synthesis and biological evaluation of biotinylated ZJ-101.

ZJ-101 is a structurally simplified analog of marine natural product superstolide A that was previously designed and synthesized in our laboratory. Biological investigation shows that ZJ-101 maintains the potent anticancer activity of the original natural product with an undefined mechanism of action. To facilitate chemical biology study, a biotinylated ZJ-101 was synthesized and biologically evaluated.

Modulation of the Endomembrane System by the Anticancer Natural Product Superstolide/ZJ-101.

Marine natural products represent a unique source for clinically relevant drugs due to their vast molecular and mechanistic diversity. ZJ-101 is a structurally simplified analog of the marine natural product superstolide A, isolated from the New Caledonian sea sponge Neosiphonia Superstes. The mechanistic activity of the superstolides has until recently remained a mystery. Here, we have identified potent antiproliferative and antiadhesive effects of ZJ-101 on cancer cell lines. Furthermore, through dose-response transcriptomics, we found unique dysregulation of the endomembrane system by ZJ-101 including a selective inhibition of O-glycosylation via lectin and glycomics analysis. We applied this mechanism to a triple-negative breast cancer spheroid model and identified a potential for the reversal of 3D-induced chemoresistance, suggesting a potential for ZJ-101 as a synergistic therapeutic agent.

robustica: customizable robust independent component analysis.

BACKGROUND: Independent Component Analysis (ICA) allows the dissection of omic datasets into modules that help to interpret global molecular signatures. The inherent randomness of this algorithm can be overcome by clustering many iterations of ICA together to obtain robust components. Existing algorithms for robust ICA are dependent on the choice of clustering method and on computing a potentially biased and large Pearson distance matrix. RESULTS: We present robustica, a Python-based package to compute robust independent components with a fully customizable clustering algorithm and distance metric. Here, we exploited its customizability to revisit and optimize robust ICA systematically. Of the 6 popular clustering algorithms considered, DBSCAN performed the best at clustering independent components across ICA iterations. To enable using Euclidean distances, we created a subroutine that infers and corrects the components' signs across ICA iterations. Our subroutine increased the resolution, robustness, and computational efficiency of the algorithm. Finally, we show the applicability of robustica by dissecting over 500 tumor samples from low-grade glioma (LGG) patients, where we define two new gene expression modules with key modulators of tumor progression upon IDH1 and TP53 mutagenesis. CONCLUSION: robustica brings precise, efficient, and customizable robust ICA into the Python toolbox. Through its customizability, we explored how different clustering algorithms and distance metrics can further optimize robust ICA. Then, we showcased how robustica can be used to discover gene modules associated with combinations of features of biological interest. Taken together, given the broad applicability of ICA for omic data analysis, we envision robustica will facilitate the seamless computation and integration of robust independent components in large pipelines.

Specialization of the photoreceptor transcriptome by Srrm3-dependent microexons is required for outer segment maintenance and vision.

Retinal photoreceptors have a distinct transcriptomic profile compared to other neuronal subtypes, likely reflecting their unique cellular morphology and function in the detection of light stimuli by way of the ciliary outer segment. We discovered a layer of this molecular specialization by revealing that the vertebrate retina expresses the largest number of tissue-enriched microexons of all tissue types. A subset of these microexons is included exclusively in photoreceptor transcripts, particularly in genes involved in cilia biogenesis and vesicle-mediated transport. This microexon program is regulated by Srrm3, a paralog of the neural microexon regulator Srrm4. Despite the fact that both proteins positively regulate retina microexons in vitro, only Srrm3 is highly expressed in mature photoreceptors. Its deletion in zebrafish results in widespread down-regulation of microexon inclusion from early developmental stages, followed by other transcriptomic alterations, severe photoreceptor defects, and blindness. These results shed light on the transcriptomic specialization and functionality of photoreceptors, uncovering unique cell type-specific roles for Srrm3 and microexons with implications for retinal diseases.

Silencing of SRRM4 suppresses microexon inclusion and promotes tumor growth across cancers.

RNA splicing is widely dysregulated in cancer, frequently due to altered expression or activity of splicing factors (SFs). Microexons are extremely small exons (3-27 nucleotides long) that are highly evolutionarily conserved and play critical roles in promoting neuronal differentiation and development. Inclusion of microexons in mRNA transcripts is mediated by the SF Serine/Arginine Repetitive Matrix 4 (SRRM4), whose expression is largely restricted to neural tissues. However, microexons have been largely overlooked in prior analyses of splicing in cancer, as their small size necessitates specialized computational approaches for their detection. Here, we demonstrate that despite having low expression in normal nonneural tissues, SRRM4 is further silenced in tumors, resulting in the suppression of normal microexon inclusion. Remarkably, SRRM4 is the most consistently silenced SF across all tumor types analyzed, implying a general advantage of microexon down-regulation in cancer independent of its tissue of origin. We show that this silencing is favorable for tumor growth, as decreased SRRM4 expression in tumors is correlated with an increase in mitotic gene expression, and up-regulation of SRRM4 in cancer cell lines dose-dependently inhibits proliferation in vitro and in a mouse xenograft model. Further, this proliferation inhibition is accompanied by induction of neural-like expression and splicing patterns in cancer cells, suggesting that SRRM4 expression shifts the cell state away from proliferation and toward differentiation. We therefore conclude that SRRM4 acts as a proliferation brake, and tumors gain a selective advantage by cutting off this brake.

Design and Synthesis of Tetrazole- and Pyridine-Containing Itraconazole Analogs as Potent Angiogenesis Inhibitors.

Itraconazole, a widely used antifungal drug, was found to possess antiangiogenic activity and is currently undergoing multiple clinical trials for the treatment of different types of cancer. However, it suffers from extremely low solubility and strong interactions with many drugs through inhibition of CYP3A4, limiting its potential as a new antiangiogenic and anticancer drug. To address these issues, a series of analogs in which the phenyl group is replaced with pyridine or fluorine-substituted benzene was synthesized. Among them the pyridine- and tetrazole-containing compound 24 has significantly improved solubility and reduced CYP3A4 inhibition compared to itraconazole. Similar to itraconazole, compound 24 inhibited the AMPK/mTOR signaling axis and the glycosylation of VEGFR2. It also induced cholesterol accumulation in the endolysosome and demonstrated binding to the sterol-sensing domain of NPC1 in a simulation study. These results suggested that compound 24 may serve as an attractive candidate for the development of a new generation of antiangiogenic drug.

ProteinFishing: a protein complex generator within the ModelX toolsuite.

SUMMARY: Accurate 3D modelling of protein-protein interactions (PPI) is essential to compensate for the absence of experimentally determined complex structures. Here, we present a new set of commands within the ModelX toolsuite capable of generating atomic-level protein complexes suitable for interface design. Among these commands, the new tool ProteinFishing proposes known and/or putative alternative 3D PPI for a given protein complex. The algorithm exploits backbone compatibility of protein fragments to generate mutually exclusive protein interfaces that are quickly evaluated with a knowledge-based statistical force field. Using interleukin-10-R2 co-crystalized with interferon-lambda-3, and a database of X-ray structures containing interleukin-10, this algorithm was able to generate interleukin-10-R2/interleukin-10 structural models in agreement with experimental data. AVAILABILITY AND IMPLEMENTATION: ProteinFishing is a portable command-line tool included in the ModelX toolsuite, written in C++, that makes use of an SQL (tested for MySQL and MariaDB) relational database delivered with a template SQL dump called FishXDB. FishXDB contains the empty tables of ModelX fragments and the data used by the embedded statistical force field. ProteinFishing is compiled for Linux-64bit, MacOS-64bit and Windows-32bit operating systems. This software is a proprietary license and is distributed as an executable with its correspondent database dumps. It can be downloaded publicly at http://modelx.crg.es/. Licenses are freely available for academic users after registration on the website and are available under commercial license for for-profit organizations or companies. CONTACT: javier.delgado@crg.eu or luis.serrano@crg.eu. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Clofoctol and sorafenib inhibit prostate cancer growth via synergistic induction of endoplasmic reticulum stress and UPR pathways.

BACKGROUND/PURPOSE: Prostate cancer is a major burden on public health and a major cause of morbidity and mortality among men worldwide. Drug combination therapy is known as a powerful tool for the treatment of cancer. The aim of this study is to evaluate the synergistic inhibitory mechanisms of clofoctol and sorafenib in the treatment of prostate cancer. However, the molecular mechanisms of this phenomenon have not been illuminated clearly. In this study, we investigated the anti-tumor effects of clofoctol in combination with sorafenib in vitro and in vivo. METHODS: The activity and mechanism of clofoctol in combination with sorafenib were examined in PC-3cells. mRNA and protein expression of key players in the ER stress pathway were detected with RT-PCR and Western blotting. Cell viability was estimated by CCK-8 assay or Alamar blue assay, and apoptosis and cell cycle were monitored and measured by flow cytometry. PC-3 cells were inoculated subcutaneously in male BALB/c nude mice. The therapeutic regimen was initiated when the tumor began showing signs of growth and treatment continued for 5 weeks. RESULTS: Our data indicate that clofototol and sorafenib induce cell death through synergistic induction of endoplasmic reticulum (ER) stress, resulting in activation of the unfolded protein response (UPR). Combination therapy with clofoctol and sorafenib induced an upregulation of markers of all three ER stress pathways: PERK, IRE1 and ATF6. In addition, combination therapy with clofoctol and sorafenib markedly inhibited the growth of prostate cancer xenograft tumors, compared with clofoctol or sorafenib alone. CONCLUSION: The combination of clofoctol and sorafenib can serve as a novel clinical treatment regimen, potentially enhancing antitumor efficacy in prostate cancer and decreasing the dose and adverse effects of either clofoctol or sorafenib alone. These results lay the foundation for subsequent research on this novel therapeutic regimen in human prostate cancer.

Novel Tetrazole-Containing Analogues of Itraconazole as Potent Antiangiogenic Agents with Reduced Cytochrome P450 3A4 Inhibition.

Itraconazole has been found to possess potent antiangiogenic activity, exhibiting promising antitumor activity in several human clinical studies. The wider use of itraconazole in the treatment of cancer, however, has been limited by its potent inhibition of the drug metabolizing enzyme cytochrome P450 3A4 (CYP3A4). In an effort to eliminate the CYP3A4 inhibition while retaining its antiangiogenic activity, we designed and synthesized a series of derivatives in which the 1,2,4-triazole ring is replaced with various azoles and nonazoles. Among these analogues, 15n with tetrazole in place of 1,2,4-triazole exhibited optimal inhibition of human umbilical vein endothelial cell proliferation with an IC50 of 73 nM without a significant effect on CYP3A4 (EC50 > 20 µM). Similar to itraconazole, 15n induced Niemann-Pick C phenotype (NPC phenotype) and blocked AMPK/mechanistic target of rapamycin signaling. These results suggest that 15n is a promising angiogenesis inhibitor that can be used in combination with most other known anticancer drugs.

Astemizole Inhibits mTOR Signaling and Angiogenesis by Blocking Cholesterol Trafficking.

Cholesterol plays a key role in membrane protein function and signaling in endothelial cells. Thus, disturbing cholesterol trafficking is an effective approach for inhibiting angiogenesis. We recently identified astemizole (AST), an antihistamine drug, as a cholesterol trafficking inhibitor from a phenotypic screen. In this study, we found that AST induced cholesterol accumulation in the lysosome by binding to the sterol-sensing domain of Niemann-Pick disease, type C1 (NPC1), a lysosomal surface protein responsible for cholesterol transport. Inhibition of cholesterol trafficking by AST led to the depletion of membrane cholesterol, causing SREBP1 nuclear localization. The depletion of membrane cholesterol resulted in dissociation of mammalian target of rapamycin (mTOR) from the lysosomal surface and inactivation of mTOR signaling. These effects were effectively rescued by addition of exogenous cholesterol. AST inhibited endothelial cell proliferation, migration and tube formation in a cholesterol-dependent manner. Furthermore, AST inhibited zebrafish angiogenesis in a cholesterol-dependent manner. Together, our data suggest that AST is a new class of NPC1 antagonist that inhibits cholesterol trafficking in endothelial cells and angiogenesis.

Structure-activity relationship study of itraconazole, a broad-range inhibitor of picornavirus replication that targets oxysterol-binding protein (OSBP).

Itraconazole (ITZ) is a well-known, FDA-approved antifungal drug that is also in clinical trials for its anticancer activity. ITZ exerts its anticancer activity through several disparate targets and pathways. ITZ inhibits angiogenesis by hampering the functioning of the vascular endothelial growth receptor 2 (VEGFR2) and by indirectly inhibiting mTOR signaling. Furthermore, ITZ directly inhibits the growth of several types of tumor cells by antagonizing Hedgehog signaling. Recently, we reported that ITZ also has broad-spectrum antiviral activity against enteroviruses, cardioviruses and hepatitis C virus, independent of established ITZ-activities but instead via a novel target, oxysterol-binding protein (OSBP), a cellular lipid shuttling protein. In this study, we analyzed which structural features of ITZ are important for the OSBP-mediated antiviral activity. The backbone structure, consisting of five rings, and the sec-butyl chain are important for antiviral activity, whereas the triazole moiety, which is critical for antifungal activity, is not. The features required for OSBP-mediated antiviral activity of ITZ overlap mostly with published features required for inhibition of VEGFR2 trafficking, but not Hh signaling. Furthermore, we use in silico studies to explore how ITZ could bind to OSBP. Our data show that several pharmacological activities of ITZ can be uncoupled, which is a critical step in the development of ITZ-based antiviral compounds with greater specificity and reduced off-target effects.

Pharmacological blockade of cholesterol trafficking by cepharanthine in endothelial cells suppresses angiogenesis and tumor growth.

Cholesterol is an important modulator of membrane protein function and signaling in endothelial cells, thus making it an emerging target for anti-angiogenic agents. In this study, we employed a phenotypic screen that detects intracellular cholesterol distribution in endothelial cells (HUVEC) and identified 13 existing drugs as cholesterol trafficking inhibitors. Cepharanthine, an approved drug for anti-inflammatory and cancer management use, was amongst the candidates, which was selected for in-depth mechanistic studies to link cholesterol trafficking and angiogenesis. Cepharanthine inhibited the endolysosomal trafficking of free-cholesterol and low-density lipoprotein in HUVEC by binding to Niemann-Pick disease, type C1 (NPC1) protein and increasing the lysosomal pH. The blockade of cholesterol trafficking led to a cholesterol-dependent dissociation of mTOR from the lysosomes and inhibition of its downstream signaling. Cepharanthine inhibited angiogenesis in HUVEC and in zebrafish in a cholesterol-dependent manner. Furthermore, cepharanthine suppressed tumor growth in vivo by inhibiting angiogenesis and it enhanced the antitumor activity of the standard chemotherapy cisplatin in lung and breast cancer xenografts in mice. Altogether, these results strongly support the idea that cholesterol trafficking is a viable drug target for anti-angiogenesis and that the inhibitors identified among existing drugs, such as cepharanthine, could be potential anti-angiogenic and antitumor agents.

Simultaneous Targeting of NPC1 and VDAC1 by Itraconazole Leads to Synergistic Inhibition of mTOR Signaling and Angiogenesis.

The antifungal drug itraconazole was recently found to exhibit potent antiangiogenic activity and has since been repurposed as an investigational anticancer agent. Itraconazole has been shown to exert its antiangiogenic activity through inhibition of the mTOR signaling pathway, but the molecular mechanism of action was unknown. We recently identified the mitochondrial protein VDAC1 as a target of itraconazole and a mediator of its activation of AMPK, an upstream regulator of mTOR. However, VDAC1 could not account for the previously reported inhibition of cholesterol trafficking by itraconazole, which was also demonstrated to lead to mTOR inhibition. In this study, we demonstrate that cholesterol trafficking inhibition by itraconazole is due to direct inhibition of the lysosomal protein NPC1. We further map the binding site of itraconazole to the sterol-sensing domain of NPC1 using mutagenesis, competition with U18666A, and molecular docking. Finally, we demonstrate that simultaneous AMPK activation and cholesterol trafficking inhibition leads to synergistic inhibition of mTOR, endothelial cell proliferation, and angiogenesis.

Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling.

Identifying the molecular target(s) of small molecules is a challenging but necessary step towards understanding their mechanism of action. While several target identification methods have been developed and used to successfully elucidate the binding proteins of a variety of small molecules, these techniques have drawbacks that make them unsuitable for detecting certain types of small molecule-target interactions. In particular, non-covalent interactions that depend on native cellular conditions, such as those of membrane proteins whose structures may be perturbed upon cell lysis, are often not amenable to affinity-based target identification methods. Here, we demonstrate a method wherein a probe containing a photolabile group is used to covalently crosslink to the small molecule binding protein within the environment of the live cell, allowing the detection and isolation of the target protein without the need for maintenance of the interaction after cell lysis. This technique is a valuable tool for studying biologically interesting small molecules with unknown mechanisms, both in the context of basic biology as well as drug discovery.

Insights into the structure-activity relationship of the anticancer compound ZJ-101, a derivative of marine natural product superstolide A: A role played by the lactone moiety.

Compound ZJ-101, a structurally simplified analog of the marine natural product superstolide A, was previously developed in our laboratory. In the subsequent structure-activity relationship study, a new analog ZJ-109 was designed and synthesized to probe the importance of the lactone moiety of the molecule by replacing the lactone in ZJ-101 with a lactam. The biological evaluation showed that ZJ-109 is about 8-12 times less active against cancer cells in vitro than ZJ-101, suggesting that the lactone moiety of the molecule is important for its anticancer activity.

Targeted Delivery and Sustained Antitumor Activity of Triptolide through Glucose Conjugation.

Triptolide, a key ingredient from the traditional Chinese medicinal plant thunder god vine, which has been used to treat inflammation and autoimmune diseases for centuries, has been shown to be an irreversible inhibitor of the XPB subunit of the transcription factor TFIIH and initiation of RNA polymerase II mediated transcription. The clinical development of triptolide over the past two decades has been limited by its toxicity and low water solubility. Herein, we report the development of a glucose conjugate of triptolide, named glutriptolide, which was intended to target tumor cells overexpressing glucose transporters selectively. Glutriptolide did not inhibit XPB activity in vitro but demonstrated significantly higher cytotoxicity against tumor cells over normal cells with greater water solubility than triptolide. Furthermore, it exhibited remarkable tumor control in vivo, which is likely due to sustained stepwise release of active triptolide within cancer cells. These findings indicate that glutriptolide may serve as a promising lead for developing a new mechanistic class of anticancer drugs.

Insights into the structure-activity relationship of the anticancer compound ZJ-101, a derivative of marine natural product superstolide A: A critical role played by the conjugated trienyl lactone moiety.

Compound ZJ-101, a structurally simplified analog of the marine natural product superstolide A, was previously developed in our laboratory. In the subsequent structure-activity relationship study, two new analogs, ZJ-105 and ZJ-106, were designed and synthesized to probe the importance of the conjugated trienyl lactone moiety of the molecule by replacing the C2-C3 double bond in ZJ-101 with a single bond and switching the geometry of the C4-C5 double bond in ZJ-101 from Z to E, respectively. Biological evaluation showed that ZJ-105 completely loses antiproliferative activity whereas ZJ-106 is significantly less active against cancer cells in vitro than ZJ-101, suggesting that the conjugated trienyl lactone moiety of the molecule is critical for its anticancer activity.

Insights into the structure-activity relationship of the anticancer compound ZJ-101: A critical role played by the cyclohexene ring.

Compound ZJ-101, a structurally simplified analog of the marine natural product superstolide A, was previously developed in our laboratory. In the subsequent structure-activity relationship study, a new analog ZJ-102 was designed and synthesized to probe the importance of the cyclohexenyl group through its replacement to a phenyl group using a concise and convergent synthetic approach. The biological evaluation showed that this new analog ZJ-102 is significantly less active against cancer cells in vitro than ZJ-101, suggesting that the cyclohexenyl ring (along with its two stereogenic centers) present in ZJ-101 is important for its anticancer activity.