A High-Throughput Platform to Identify Small-Molecule Inhibitors of CRISPR-Cas9.

The precise control of CRISPR-Cas9 activity is required for a number of genome engineering technologies. Here, we report a generalizable platform that provided the first synthetic small-molecule inhibitors of Streptococcus pyogenes Cas9 (SpCas9) that weigh <500 Da and are cell permeable, reversible, and stable under physiological conditions. We developed a suite of high-throughput assays for SpCas9 functions, including a primary screening assay for SpCas9 binding to the protospacer adjacent motif, and used these assays to screen a structurally diverse collection of natural-product-like small molecules to ultimately identify compounds that disrupt the SpCas9-DNA interaction. Using these synthetic anti-CRISPR small molecules, we demonstrated dose and temporal control of SpCas9 and catalytically impaired SpCas9 technologies, including transcription activation, and identified a pharmacophore for SpCas9 inhibition using structure-activity relationships. These studies establish a platform for rapidly identifying synthetic, miniature, cell-permeable, and reversible inhibitors against both SpCas9 and next-generation CRISPR-associated nucleases.

Selective Inhibition of FOXO1 Activator/Repressor Balance Modulates Hepatic Glucose Handling.

Insulin resistance is a hallmark of diabetes and an unmet clinical need. Insulin inhibits hepatic glucose production and promotes lipogenesis by suppressing FOXO1-dependent activation of G6pase and inhibition of glucokinase, respectively. The tight coupling of these events poses a dual conundrum: mechanistically, as the FOXO1 corepressor of glucokinase is unknown, and clinically, as inhibition of glucose production is predicted to increase lipogenesis. Here, we report that SIN3A is the insulin-sensitive FOXO1 corepressor of glucokinase. Genetic ablation of SIN3A abolishes nutrient regulation of glucokinase without affecting other FOXO1 target genes and lowers glycemia without concurrent steatosis. To extend this work, we executed a small-molecule screen and discovered selective inhibitors of FOXO-dependent glucose production devoid of lipogenic activity in hepatocytes. In addition to identifying a novel mode of insulin action, these data raise the possibility of developing selective modulators of unliganded transcription factors to dial out adverse effects of insulin sensitizers.

Structure, mechanism, and inhibition of Hedgehog acyltransferase.

The Sonic Hedgehog (SHH) morphogen pathway is fundamental for embryonic development and stem cell maintenance and is implicated in various cancers. A key step in signaling is transfer of a palmitate group to the SHH N terminus, catalyzed by the multi-pass transmembrane enzyme Hedgehog acyltransferase (HHAT). We present the high-resolution cryo-EM structure of HHAT bound to substrate analog palmityl-coenzyme A and a SHH-mimetic megabody, revealing a heme group bound to HHAT that is essential for HHAT function. A structure of HHAT bound to potent small-molecule inhibitor IMP-1575 revealed conformational changes in the active site that occlude substrate binding. Our multidisciplinary analysis provides a detailed view of the mechanism by which HHAT adapts the membrane environment to transfer an acyl chain across the endoplasmic reticulum membrane. This structure of a membrane-bound O-acyltransferase (MBOAT) superfamily member provides a blueprint for other protein-substrate MBOATs and a template for future drug discovery.

Synthetic Lethal and Resistance Interactions with BET Bromodomain Inhibitors in Triple-Negative Breast Cancer.

BET bromodomain inhibitors (BBDIs) are candidate therapeutic agents for triple-negative breast cancer (TNBC) and other cancer types, but inherent and acquired resistance to BBDIs limits their potential clinical use. Using CRISPR and small-molecule inhibitor screens combined with comprehensive molecular profiling of BBDI response and resistance, we identified synthetic lethal interactions with BBDIs and genes that, when deleted, confer resistance. We observed synergy with regulators of cell cycle progression, YAP, AXL, and SRC signaling, and chemotherapeutic agents. We also uncovered functional similarities and differences among BRD2, BRD4, and BRD7. Although deletion of BRD2 enhances sensitivity to BBDIs, BRD7 loss leads to gain of TEAD-YAP chromatin binding and luminal features associated with BBDI resistance. Single-cell RNA-seq, ATAC-seq, and cellular barcoding analysis of BBDI responses in sensitive and resistant cell lines highlight significant heterogeneity among samples and demonstrate that BBDI resistance can be pre-existing or acquired.

Targeted chemotherapy overcomes drug resistance in melanoma.

The emergence of drug resistance is a major obstacle for the success of targeted therapy in melanoma. Additionally, conventional chemotherapy has not been effective as drug-resistant cells escape lethal DNA damage effects by inducing growth arrest commonly referred to as cellular dormancy. We present a therapeutic strategy termed "targeted chemotherapy" by depleting protein phosphatase 2A (PP2A) or its inhibition using a small molecule inhibitor (1,10-phenanthroline-5,6-dione [phendione]) in drug-resistant melanoma. Targeted chemotherapy induces the DNA damage response without causing DNA breaks or allowing cellular dormancy. Phendione treatment reduces tumor growth of BRAFV600E-driven melanoma patient-derived xenografts (PDX) and diminishes growth of NRASQ61R-driven melanoma, a cancer with no effective therapy. Remarkably, phendione treatment inhibits the acquisition of resistance to BRAF inhibition in BRAFV600E PDX highlighting its effectiveness in combating the advent of drug resistance.

Focal adhesion kinase signaling - tumor vulnerabilities and clinical opportunities.

Focal adhesion kinase (FAK; encoded by PTK2) was discovered over 30 years ago as a cytoplasmic protein tyrosine kinase that is localized to cell adhesion sites, where it is activated by integrin receptor binding to extracellular matrix proteins. FAK is ubiquitously expressed and functions as a signaling scaffold for a variety of proteins at adhesions and in the cell cytoplasm, and with transcription factors in the nucleus. FAK expression and intrinsic activity are essential for mouse development, with molecular connections to cell motility, cell survival and gene expression. Notably, elevated FAK tyrosine phosphorylation is common in tumors, including pancreatic and ovarian cancers, where it is associated with decreased survival. Small molecule and orally available FAK inhibitors show on-target inhibition in tumor and stromal cells with effects on chemotherapy resistance, stromal fibrosis and tumor microenvironment immune function. Herein, we discuss recent insights regarding mechanisms of FAK activation and signaling, its roles as a cytoplasmic and nuclear scaffold, and the tumor-intrinsic and -extrinsic effects of FAK inhibitors. We also discuss results from ongoing and advanced clinical trials targeting FAK in low- and high-grade serous ovarian cancers, where FAK acts as a master regulator of drug resistance. Although FAK is not known to be mutationally activated, preventing FAK activity has revealed multiple tumor vulnerabilities that support expanding clinical combinatorial targeting possibilities.

High-resolution RNA tertiary structures in Zika virus stem-loop A for the development of inhibitory small molecules.

Flaviviruses such as Zika (ZIKV) and dengue virus (DENV) are positive-sense RNA viruses belonging to Flaviviridae The flavivirus genome contains a 5' end stem-loop promoter sequence known as stem-loop A (SLA) that is recognized by the flavivirus polymerase NS5 during viral RNA synthesis and 5' guanosine cap methylation. The crystal structures of ZIKV and DENV SLAs show a well-defined fold, consisting of a bottom stem, side loop, and top stem-loop, providing unique interaction sites for small molecule inhibitors to disrupt the promoter function. To facilitate the identification of small molecule binding sites in flavivirus SLA, we determined high-resolution structures of the bottom and top stems of ZIKV SLA, which contain a single U- or G-bulge, respectively. Both bulge nucleotides exhibit multiple orientations, from folded back on the adjacent nucleotide to flipped out of the helix, and are stabilized by stacking or base triple interactions. These structures suggest that even a single unpaired nucleotide can provide flexibility to RNA structures, and its conformation is mainly determined by the stabilizing chemical environment. To facilitate discovery of small molecule inhibitors that interfere with the functions of ZIKV SLA, we screened and identified compounds that bind to the bottom and top stems of ZIKV SLA.

Targeting STING oligomerization with small-molecule inhibitors.

Stimulator of interferon genes (STING) is an essential adaptor protein required for the inflammatory response to cytosolic DNA. dsDNA activates cGAS to generate cGAMP, which binds and activates STING triggering a conformational change, oligomerization, and the IRF3- and NFκB-dependent transcription of type I Interferons (IFNs) and inflammatory cytokines, as well as the activation of autophagy. Aberrant activation of STING is now linked to a growing number of both rare as well as common chronic inflammatory diseases. Here, we identify and characterize a potent small-molecule inhibitor of STING. This compound, BB-Cl-amidine inhibits STING signaling and production of type I IFNs, IFN-stimulated genes (ISGs) and NFκB-dependent cytokines, but not other pattern recognition receptors. In vivo, BB-Cl-amidine alleviated pathology resulting from accrual of cytosolic DNA in Trex-1 mutant mice. Mechanistically BB-Cl-amidine inhibited STING oligomerization through modification of Cys148. Collectively, our work uncovers an approach to inhibit STING activation and highlights the potential of this strategy for the treatment of STING-driven inflammatory diseases.

Revealing the mechanism of action of a first-in-class covalent inhibitor of KRASG12C (ON) and other functional properties of oncogenic KRAS by 31P NMR.

Individual oncogenic KRAS mutants confer distinct differences in biochemical properties and signaling for reasons that are not well understood. KRAS activity is closely coupled to protein dynamics and is regulated through two interconverting conformations: state 1 (inactive, effector binding deficient) and state 2 (active, effector binding enabled). Here, we use 31P NMR to delineate the differences in state 1 and state 2 populations present in WT and common KRAS oncogenic mutants (G12C, G12D, G12V, G13D, and Q61L) bound to its natural substrate GTP or a commonly used nonhydrolyzable analog GppNHp (guanosine-5'-[(ß,γ)-imido] triphosphate). Our results show that GppNHp-bound proteins exhibit significant state 1 population, whereas GTP-bound KRAS is primarily (90% or more) in state 2 conformation. This observation suggests that the predominance of state 1 shown here and in other studies is related to GppNHp and is most likely nonexistent in cells. We characterize the impact of this differential conformational equilibrium of oncogenic KRAS on RAF1 kinase effector RAS-binding domain and intrinsic hydrolysis. Through a KRAS G12C drug discovery, we have identified a novel small-molecule inhibitor, BBO-8956, which is effective against both GDP- and GTP-bound KRAS G12C. We show that binding of this inhibitor significantly perturbs state 1-state 2 equilibrium and induces an inactive state 1 conformation in GTP-bound KRAS G12C. In the presence of BBO-8956, RAF1-RAS-binding domain is unable to induce a signaling competent state 2 conformation within the ternary complex, demonstrating the mechanism of action for this novel and active-conformation inhibitor.

Small-molecule Ro-08-2750 interacts with many RNA-binding proteins and elicits MUSASHI2-independent phenotypes.

RNA-binding proteins (RBPs) are key regulators of gene expression. Small molecules targeting these RBP-RNA interactions are a rapidly emerging class of therapeutics for treating a variety of diseases. Ro-08-2750 (Ro) is a small molecule identified as a competitive inhibitor of Musashi (MSI)-RNA interactions. Here, we show that multiple Ro-dependent cellular phenotypes, specifically adrenocortical steroid production and cell viability, are Musashi-2 (MSI2)-independent. Using an unbiased proteome-wide approach, we discovered Ro broadly interacts with RBPs, many containing RRM domains. To confirm this finding, we leveraged the large-scale ENCODE data to identify a subset of RBPs whose depletion phenocopies Ro inhibition, indicating Ro is a promiscuous inhibitor of multiple RBPs. Consistent with broad disruption of ribonucleoprotein complexes, Ro treatment leads to stress granule formation. This strategy represents a generalizable framework for validating the specificity and identifying targets of RBP inhibitors in a cellular context.

A specific JMJD6 inhibitor potently suppresses multiple types of cancers both in vitro and in vivo.

Jumonji C-domain-containing protein 6 (JMJD6), an iron (Fe2+) and α-ketoglutarate (α-KG)-dependent oxygenase, is expressed at high levels, correlated with poor prognosis, and considered as a therapeutic target in multiple cancer types. However, specific JMJD6 inhibitors that are potent in suppressing tumorigenesis have not been reported so far. We herein report that iJMJD6, a specific small-molecule inhibitor of JMJD6 with favorable physiochemical properties, inhibits the enzymatic activity of JMJD6 protein both in vitro and in cultured cells. iJMJD6 is effective in suppressing cell proliferation, migration, and invasion in multiple types of cancer cells in a JMJD6-dependent manner, while it exhibits minimal toxicity in normal cells. Mechanistically, iJMJD6 represses the expression of oncogenes, including Myc and CCND1, in accordance with JMJD6 function in promoting the transcription of these genes. iJMJD6 exhibits suitable pharmacokinetic properties and suppresses tumor growth in multiple cancer cell line- and patient-derived xenograft models safely. Furthermore, combination therapy with iJMJD6 and BET protein inhibitor (BETi) JQ1 or estrogen receptor antagonist fulvestrant exhibits synergistic effects in suppressing tumor growth. Taken together, we demonstrate that inhibition of JMJD6 enzymatic activity by using iJMJD6 is effective in suppressing oncogene expression and cancer development, providing a therapeutic avenue for treating cancers that are dependent on JMJD6 in the clinic.

Increased Siglec-9/Siglec-9L interactions on NK cells predict poor HCC prognosis and present a targetable checkpoint for immunotherapy.

BACKGROUND & AIMS: Natural killer (NK) cell-based anti-hepatocellular carcinoma (HCC) therapy is an increasingly attractive approach that warrants further study. Siglec-9 interacts with its ligand (Siglec-9L) and restrains NK cell functions, suggesting it is a potential therapeutic target. However, in situ Siglec-9/Siglec-9L interactions in HCC have not been reported, and a relevant interventional strategy is lacking. Herein, we aim to illustrate Siglec-9/Siglec-9L-mediated cell sociology and identify small-molecule inhibitors targeting Siglec-9 that could improve the efficacy of NK cell-based immunotherapy for HCC. METHODS: Multiplexed immunofluorescence staining was performed to analyze the expression pattern of Siglec-7, -9 and their ligands in HCC tissues. Then we conducted docking-based virtual screening combined with bio-layer interferometry assays to identify a potent small-molecule Siglec-9 inhibitor. The therapeutic potential was further evaluated in vitro and in hepatoma-bearing NCG mice. RESULTS: Siglec-9 expression, rather than Siglec-7, was markedly upregulated on tumor-infiltrating NK cells, which correlated significantly with reduced survival of patients with HCC. Moreover, the number of Siglec-9L+ cells neighboring Siglec-9+ NK cells was increased in HCC tissues and was also associated with tumor recurrence and reduced survival, further suggesting that Siglec-9/Siglec-9L interactions are a potential therapeutic target in HCC. In addition, we identified a small-molecule Siglec-9 inhibitor MTX-3937 which inhibited phosphorylation of Siglec-9 and downstream SHP1 and SHP2. Accordingly, MTX-3937 led to considerable improvement in NK cell function. Notably, MTX-3937 enhanced cytotoxicity of both human peripheral and tumor-infiltrating NK cells. Furthermore, transfer of MTX-3937-treated NK92 cells greatly suppressed the growth of hepatoma xenografts in NCG mice. CONCLUSIONS: Our study provides the rationale for HCC treatment by targeting Siglec-9 on NK cells and identifies a promising small-molecule inhibitor against Siglec-9 that enhances NK cell-mediated HCC surveillance. IMPACT AND IMPLICATIONS: Herein, we found that Siglec-9 expression is markedly upregulated on tumor-infiltrating natural killer (TINK) cells and correlates with reduced survival in patients with hepatocellular carcinoma (HCC). Moreover, the number of Siglec-9L+ cells neighboring Siglec-9+ NK cells was increased in HCC tissues and was also associated with tumor recurrence and reduced survival. More importantly, we identified a small-molecule inhibitor targeting Siglec-9 that augments NK cell functions, revealing a novel immunotherapy strategy for liver cancer that warrants further clinical investigation.

Mechanism and inhibition of the papain-like protease, PLpro, of SARS-CoV-2.

The SARS-CoV-2 coronavirus encodes an essential papain-like protease domain as part of its non-structural protein (nsp)-3, namely SARS2 PLpro, that cleaves the viral polyprotein, but also removes ubiquitin-like ISG15 protein modifications as well as, with lower activity, Lys48-linked polyubiquitin. Structures of PLpro bound to ubiquitin and ISG15 reveal that the S1 ubiquitin-binding site is responsible for high ISG15 activity, while the S2 binding site provides Lys48 chain specificity and cleavage efficiency. To identify PLpro inhibitors in a repurposing approach, screening of 3,727 unique approved drugs and clinical compounds against SARS2 PLpro identified no compounds that inhibited PLpro consistently or that could be validated in counterscreens. More promisingly, non-covalent small molecule SARS PLpro inhibitors also target SARS2 PLpro, prevent self-processing of nsp3 in cells and display high potency and excellent antiviral activity in a SARS-CoV-2 infection model.

A phase 1/1b, open-label, dose-escalation study of PD-1 inhibitor, cetrelimab alone and in combination with FGFR inhibitor, erdafitinib in Japanese patients with advanced solid tumors.

Immune checkpoint inhibitors are the leading approaches in tumor immunotherapy. The aim of the study was to establish recommended phase 2 doses (RP2Ds) of intravenous cetrelimab, a checkpoint inhibitor, alone and with oral erdafitinib in Japanese patients with advanced solid tumors. This open-label, non-randomized, dose-escalation phase 1/1b study enrolled adults with advanced solid tumors who were ineligible for standard therapy. Study was conducted in two parts: phase 1a assessed cetrelimab at three dosing levels (80 mg every 2 weeks [Q2W], 240 mg Q2W, and 480 mg Q4W); phase 1b assessed cetrelimab+erdafitinib at two dosing levels (240 mg Q2W + 6 mg once daily [QD] and 240 mg Q2W + 8 mg QD). Primary endpoint was frequency and severity of dose-limiting toxicities (DLTs) of cetrelimab ± erdafitinib. In total 22 patients (phase 1a, n = 9; phase 1b, n = 13) were enrolled. Median duration of follow-up was 8.64 months in phase 1a and 2.33 months in phase 1b. In phase 1a, DLTs weren't reported while in phase 1b, 1 patient who received 240 mg cetrelimab + 6 mg erdafitinib reported Stevens-Johnson syndrome (grade 3, immune-related). Overall, 88.9% patients in phase 1a (grade ≥ 3: 44.4%) and 100.0% in phase 1b (grade ≥ 3: 53.8%) experienced ≥ 1 treatment-related adverse events (TEAEs); 33.3% in phase 1a and 38.5% in phase 1b reported serious TEAEs, of which 11.1% patients in phase 1a and 15.4% in phase 1b had TEAEs which led to treatment discontinuation. Cetrelimab alone and in combination with erdafitinib showed manageable safety in Japanese patients with advanced solid tumors. RP2Ds were determined as 480 mg cetrelimab Q4W for monotherapy, and cetrelimab 240 mg Q2W + erdafitinib 8 mg QD for combination therapy.

Impact of DNA ligase inhibition on the nick sealing of polß nucleotide insertion products at the downstream steps of base excision repair pathway.

DNA ligase (LIG) I and IIIα finalize base excision repair (BER) by sealing a nick product after nucleotide insertion by DNA polymerase (pol) ß at the downstream steps. We previously demonstrated that a functional interplay between polß and BER ligases is critical for efficient repair, and polß mismatch or oxidized nucleotide insertions confound final ligation step. Yet, how targeting downstream enzymes with small molecule inhibitors could affect this coordination remains unknown. Here, we report that DNA ligase inhibitors, L67 and L82-G17, slightly enhance hypersensitivity to oxidative stress-inducing agent, KBrO3, in polß+/+ cells more than polß-/- null cells. We showed less efficient ligation after polß nucleotide insertions in the presence of the DNA ligase inhibitors. Furthermore, the mutations at the ligase inhibitor binding sites (G448, R451, A455) of LIG1 significantly affect nick DNA binding affinity and nick sealing efficiency. Finally, our results demonstrated that the BER ligases seal a gap repair intermediate by the effect of polß inhibitor that diminishes gap filling activity. Overall, our results contribute to understand how the BER inhibitors against downstream enzymes, polß, LIG1, and LIGIIIα, could impact the efficiency of gap filling and subsequent nick sealing at the final steps leading to the formation of deleterious repair intermediates.

Discovery of novel macrocyclic derivatives as potent and selective cyclin-dependent kinase 2 inhibitors.

Cyclin-dependent kinase 2 (CDK2) is a member of CDK family of kinases (CDKs) that regulate the cell cycle. Its inopportune or over-activation leads to uncontrolled cell cycle progression and drives numerous types of cancers, especially ovarian, uterine, gastric cancer, as well as those associated with amplified CCNE1 gene. However, developing selective lead compound as CDK2 inhibitors remains challenging owing to similarities in the ATP pockets among different CDKs. Herein, we described the optimization of compound 1, a novel macrocyclic inhibitor targeting CDK2/5/7/9, aiming to discover more selective and metabolically stable lead compound as CDK2 inhibitor. Molecular dynamic (MD) simulations were performed for compound 1 and 9 to gain insights into the improved selectivity against CDK5. Further optimization efforts led to compound 22, exhibiting excellent CDK2 inhibitory activity, good selectivity over other CDKs and potent cellular effects. Based on these characterizations, we propose that compound 22 holds great promise as a potential lead candidate for drug development.

Small-molecule inhibitors for the Prp8 intein as antifungal agents.

Self-splicing proteins, called inteins, are present in many human pathogens, including the emerging fungal threats Cryptococcus neoformans (Cne) and Cryptococcus gattii (Cga), the causative agents of cryptococcosis. Inhibition of protein splicing in Cryptococcus sp. interferes with activity of the only intein-containing protein, Prp8, an essential intron splicing factor. Here, we screened a small-molecule library to find addititonal, potent inhibitors of the Cne Prp8 intein using a split-GFP splicing assay. This revealed the compound 6G-318S, with IC50 values in the low micromolar range in the split-GFP assay and in a complementary split-luciferase system. A fluoride derivative of the compound 6G-318S displayed improved cytotoxicity in human lung carcinoma cells, although there was a slight reduction in the inhibition of splicing. 6G-318S and its derivative inhibited splicing of the Cne Prp8 intein in vivo in Escherichia coli and in C. neoformans Moreover, the compounds repressed growth of WT C. neoformans and C. gattii In contrast, the inhibitors were less potent at inhibiting growth of the inteinless Candida albicans Drug resistance was observed when the Prp8 intein was overexpressed in C. neoformans, indicating specificity of this molecule toward the target. No off-target activity was observed, such as inhibition of serine/cysteine proteases. The inhibitors bound covalently to the Prp8 intein and binding was reduced when the active-site residue Cys1 was mutated. 6G-318S showed a synergistic effect with amphotericin B and additive to indifferent effects with a few other clinically used antimycotics. Overall, the identification of these small-molecule intein-splicing inhibitors opens up prospects for a new class of antifungals.

1-Benzylimidazole attenuates the stemness of breast cancer cells through partially targeting CYP4Z1.

Cytochrome P450 (CYP) 4Z1 (CYP4Z1) has recently garnered much interest as its expression predicts a poor prognosis and as a oncogene in breast cancer, and overexpressed in other many cancers. We previously showed that CYP4Z1 acts as a promoter of cancer stem cells (CSCs) to facilitate the occurrence and development of breast cancer. Here, RNA sequencing found that 1-benzylimidazole (1-Benzy) held a preferable correlation with breast cancer and suppressed the expression of CSC makers. Further functional experiments, including mammary spheroid formation, wound-healing, transwell-invasion, detection of tumor initiation, and metastatic ability, showed that 1-Benzy suppressed the stemness and metastasis of breast cancer cells. Additionally, we further demonstrated that CYP4Z1 is necessary for 1-Benzy-mediated suppression on breast cancer stemness and 1-Benzy exerted a weaker effect in breast cancer cells with CYP4Z1 knockdown. Taken together, our data suggest that 1-Benzy might be a potential drug suppressing breast cancer stemness via targeting CYP4Z1.

Regulating Protein-RNA Interactions: Advances in Targeting the LIN28/Let-7 Pathway.

Originally discovered in C. elegans, LIN28 is an evolutionarily conserved zinc finger RNA-binding protein (RBP) that post-transcriptionally regulates genes involved in developmental timing, stem cell programming, and oncogenesis. LIN28 acts via two distinct mechanisms. It blocks the biogenesis of the lethal-7 (let-7) microRNA (miRNA) family, and also directly binds messenger RNA (mRNA) targets, such as IGF-2 mRNA, and alters downstream splicing and translation events. This review focuses on the molecular mechanism of LIN28 repression of let-7 and current strategies to overcome this blockade for the purpose of cancer therapy. We highlight the value of the LIN28/let-7 pathway as a drug target, as multiple oncogenic proteins that the pathway regulates are considered undruggable due to their inaccessible cellular location and lack of cavities for small molecule binding.

Pentagalloyl Glucose (PGG) Exhibits Anti-Cancer Activity against Aggressive Prostate Cancer by Modulating the ROR1 Mediated AKT-GSK3ß Pathway.

Androgen-receptor-negative, androgen-independent (ARneg-AI) prostate cancer aggressively proliferates and metastasizes, which makes treatment difficult. Hence, it is necessary to continue exploring cancer-associated markers, such as oncofetal Receptor Tyrosine Kinase like Orphan Receptor 1 (ROR1), which may serve as a form of targeted prostate cancer therapy. In this study, we identify that Penta-O-galloyl-ß-D-glucose (PGG), a plant-derived gallotannin small molecule inhibitor, modulates ROR1-mediated oncogenic signaling and mitigates prostate cancer phenotypes. Results indicate that ROR1 protein levels were elevated in the highly aggressive ARneg-AI PC3 cancer cell line. PGG was selectively cytotoxic to PC3 cells and induced apoptosis of PC3 (IC50 of 31.64 µM) in comparison to normal prostate epithelial RWPE-1 cells (IC50 of 74.55 µM). PGG was found to suppress ROR1 and downstream oncogenic pathways in PC3 cells. These molecular phenomena were corroborated by reduced migration, invasion, and cell cycle progression of PC3 cells. PGG minimally and moderately affected RWPE-1 and ARneg-AI DU145, respectively, which may be due to these cells having lower levels of ROR1 expression in comparison to PC3 cells. Additionally, PGG acted synergistically with the standard chemotherapeutic agent docetaxel to lower the IC50 of both compounds about five-fold (combination index = 0.402) in PC3 cells. These results suggest that ROR1 is a key oncogenic driver and a promising target in aggressive prostate cancers that lack a targetable androgen receptor. Furthermore, PGG may be a selective and potent anti-cancer agent capable of treating ROR1-expressing prostate cancers.