Structure of a Signaling Cannabinoid Receptor 1-G Protein Complex.

Cannabis elicits its mood-enhancing and analgesic effects through the cannabinoid receptor 1 (CB1), a G protein-coupled receptor (GPCR) that signals primarily through the adenylyl cyclase-inhibiting heterotrimeric G protein Gi. Activation of CB1-Gi signaling pathways holds potential for treating a number of neurological disorders and is thus crucial to understand the mechanism of Gi activation by CB1. Here, we present the structure of the CB1-Gi signaling complex bound to the highly potent agonist MDMB-Fubinaca (FUB), a recently emerged illicit synthetic cannabinoid infused in street drugs that have been associated with numerous overdoses and fatalities. The structure illustrates how FUB stabilizes the receptor in an active state to facilitate nucleotide exchange in Gi. The results compose the structural framework to explain CB1 activation by different classes of ligands and provide insights into the G protein coupling and selectivity mechanisms adopted by the receptor.

Novel Aza-podophyllotoxin derivative induces oxidative phosphorylation and cell death via AMPK activation in triple-negative breast cancer.

BACKGROUND: To circumvent Warburg effect, several clinical trials for different cancers are utilising a combinatorial approach using metabolic reprogramming and chemotherapeutic agents including metformin. The majority of these metabolic interventions work via indirectly activating AMP-activated protein kinase (AMPK) to alter cellular metabolism in favour of oxidative phosphorylation over aerobic glycolysis. The effect of these drugs is dependent on glycaemic and insulin conditions.  Therefore, development of small molecules, which can activate AMPK, irrespective of the energy state, may be a better approach for triple-negative breast cancer (TNBC) treatment. METHODS: Therapeutic effect of SU212 on TNBC cells was examined using in vitro and in vivo models. RESULTS: We developed and characterised the efficacy of novel AMPK activator (SU212) that selectively induces oxidative phosphorylation and decreases glycolysis in TNBC cells, while not affecting these pathways in normal cells.   SU212 accomplished this metabolic reprogramming by activating AMPK independent of energy stress and irrespective of the glycaemic/insulin state. This leads to mitotic phase arrest and apoptosis in TNBC cells. In vivo, SU212 inhibits tumour growth, cancer progression and metastasis. CONCLUSIONS: SU212 directly activates AMPK in TNBC cells, but does not hamper glucose metabolism in normal cells. Our study provides compelling preclinical data for further development of SU212 for the treatment of TNBC.

Facile synthesis of C6-substituted benz[4,5]imidazo[1,2-a]quinoxaline derivatives and their anticancer evaluation.

Cancer remains a leading cause of death worldwide, resulting in continuous efforts to discover and develop highly efficacious anticancer drugs. High-throughput screening of heterocyclic compound libraries is one of the promising approaches that provided several new lead molecules with a novel mechanism of action. On the basis of the promising anticancer potential of imidazoquinoxaline as well as the structurally similar imidazoquinoline-derived scaffold, we prepared a set of C6-substituted benzimidazo[1,2-a]quinoxaline derivatives via two novel synthetic routes using commercially available starting materials, with good to excellent yields and evaluated their anticancer activity against the NCI-60 cancer cell lines. The one-dose (10 µM) anticancer screening of the synthesized compounds in the NCI-60 cell line panel revealed that the substituents have a significant role in the activity. In particular, the indole (7f), imidazole (7g), and benzimidazole (7h) derivatives showed significant activity against the triple-negative breast cancer cell line, MDA-MB-468. The lead compounds also exhibited notable IC50 values against another breast cancer cell line, MCF-7. Furthermore, it was observed that these compounds were relatively nontoxic to normal cell lines: HEK293 (human embryonic kidney cell line) and MCF12A (nontumorigenic human breast epithelial cell line). The IC50 values against healthy cells were at least 5- to 11-fold higher, offering a new class of heterocycles that can be further developed as promising therapeutics for cancer treatment.

First results from the XMCD facility at the Energy-Dispersive EXAFS beamline of the Indus-2 synchrotron source.

Setting up of the X-ray Magnetic Circular Dichroism (XMCD) measurement facility with hard X-rays at the Energy-Dispersive EXAFS beamline (BL-08) at the Indus-2 synchrotron source is reported. This includes the design and development of a water-cooled electromagnet having a highest magnetic field of 2 T in a good field volume of 125 mm3 and having a 10 mm hole throughout for passage of the synchrotron beam. This also includes the development of an (X-Z-θ) motion stage for the heavy electromagnet for aligning its axis and the beam hole along the synchrotron beam direction. Along with the above developments, also reported is the first XMCD signal measured on a thick Gd film in the above set-up which shows good agreement with the reported results. This is the first facility to carry out XMCD measurement with hard X-rays in India.

Synthetic, Structural, and Anticancer Activity Evaluation Studies on Novel Pyrazolylnucleosides.

The synthesis of novel pyrazolylnucleosides 3a-e, 4a-e, 5a-e, and 6a-e are described. The structures of the regioisomers were elucidated by using extensive NMR studies. The pyrazolylnucleosides 5a-e and 6a-e were screened for anticancer activities on sixty human tumor cell lines. The compound 6e showed good activity against 39 cancer cell lines. In particular, it showed significant inhibition against the lung cancer cell line Hop-92 (GI50 9.3 µM) and breast cancer cell line HS 578T (GI50 3.0 µM).

Quantitative Proteomic Profiling Reveals Key Pathways in the Anticancer Action of Methoxychalcone Derivatives in Triple Negative Breast Cancer.

Triple negative breast cancer is an aggressive, heterogeneous disease with high recurrence and metastasis rates even with modern chemotherapy regimens and thus is in need of new therapeutics. Here, three novel synthetic analogues of chalcones, plant-based molecules that have demonstrated potency against a wide variety of cancers, were investigated as potential therapeutics for triple negative breast cancer. These compounds exhibit IC50 values of ∼5 µM in triple negative breast cancer cell lines and are more potent against triple negative breast cancer cell lines than against nontumor breast cell lines according to viability experiments. Tandem mass tag-based quantitative proteomics followed by gene set enrichment analysis and validation experiments using flow cytometry, apoptosis, and Western blot assays revealed three different anticancer mechanisms for these compounds. First, the chalcone analogues induce the unfolded protein response followed by apoptosis. Second, increases in the abundances of MHC-I pathway proteins occurs, which would likely result in immune stimulation in an organism. And third, treatment with the chalcone analogues causes disruption of the cell cycle by interfering with microtubule structure and by inducing G1 phase arrest. These data demonstrate the potential of these novel chalcone derivatives as treatments for triple negative breast cancer, though further work evaluating their efficacy in vivo is needed.

Molecular Imaging Biosensor Monitors p53 Sumoylation in Cells and Living Mice.

Small molecule mediated stabilization of p53 tumor suppressor protein through sumoylation is a promising new strategy for improving cancer chemotherapy. A molecular tool that monitors p53 sumoylation status and expedites screening for drugs that enhance p53 sumoylation would be beneficial. We report a molecularly engineered reporter fragment complementation biosensor based on optical imaging of Firefly luciferase (FLuc), to quantitatively image p53 sumoylation and desumoylation in cells and living mice. We initially characterized this biosensor by successfully imaging sumoylation of several target proteins, achieving significant FLuc complementation for ERα (p < 0.01), p53 (p < 0.005), FKBP12 (p < 0.03), ID (p < 0.03), and HDAC1 (p < 0.002). We then rigorously tested the sensitivity and specificity of the biosensor using several variants of p53 and SUMO1, including deletion mutants, and those with modified sequences containing the SUMO-acceptor site of target proteins. Next we evaluated the performance of the biosensor in HepG2 cells by treatment with ginkgolic acid, a drug that reduces p53 sumoylation, as well as trichostatin A, a potential inducer of p53 sumoylation by enhancement of its nuclear export. Lastly, we demonstrated the in vivo utility of this biosensor in monitoring and quantifying the effects of these drugs on p53 sumoylation in living mice using bioluminescence imaging. Adoption of this biosensor in future high throughput drug screening has the important potential to help identify new and repurposed small molecules that alter p53 sumoylation, and to preclinically evaluate candidate anticancer drugs in living animals.

A role for RIO kinases in the crosshair of cancer research and therapy.

RIO (right open reading frame) family of kinases including RIOK1, RIOK2 and RIOK3 are known for their role in the ribosomal biogenesis. Dysfunction of RIO kinases have been implicated in malignancies, including acute myeloid leukemia, glioma, breast, colorectal, lung and prostatic adenocarcinoma suggesting RIO kinases as potential targets in cancer. In vitro, in vivo and clinical studies have demonstrated that RIO kinases are overexpressed in various types of cancers suggesting important roles in tumorigenesis, especially in metastasis. In the context of malignancies, RIO kinases are involved in cancer-promoting pathways including AKT/mTOR, RAS, p53 and NF-κB and cell cycle regulation. Here we review the role of RIO kinases in cancer development emphasizing their potential as therapeutic target and encouraging further development and investigation of inhibitors in the context of cancer.

Inhibition of protein translational machinery in triple-negative breast cancer as a promising therapeutic strategy.

Y-box binding protein-1 (YB-1) is a proto-oncogenic protein associated with protein translation regulation. It plays a crucial role in the development and progression of triple-negative breast cancer (TNBC). In this study, we describe a promising approach to inhibit YB-1 using SU056, a small-molecule inhibitor. SU056 physically interacts with YB-1 and reduces its expression, which helps to restrain the progression of TNBC. Proteome profiling analysis indicates that the inhibition of YB-1 by SU056 can alter the proteins that regulate protein translation, an essential process for cancer cell growth. Preclinical studies on human cells, mice, and patient-derived xenograft tumor models show the effectiveness of SU056. Moreover, toxicological studies have shown that SU056 treatment and dosing are well tolerated without any adverse effects. Overall, our study provides a strong foundation for the further development of SU056 as a potential treatment option for patients with TNBC by targeting YB-1.

Mechanism of the antiproliferative activity of some naphthalene diimide G-quadruplex ligands.

G-quadruplexes are higher-order nucleic acid structures that can form in G-rich telomeres and promoter regions of oncogenes. Telomeric quadruplex stabilization by small molecules can lead to telomere uncapping, followed by DNA damage response and senescence, as well as chromosomal fusions leading to deregulation of mitosis, followed by apoptosis and downregulation of oncogene expression. We report here on investigations into the mechanism of action of tetra-substituted naphthalene diimide ligands on the basis of cell biologic data together with a National Cancer Institute COMPARE study. We conclude that four principal mechanisms of action are implicated for these compounds: 1) telomere uncapping with subsequent DNA damage response and senescence; 2) inhibition of transcription/translation of oncogenes; 3) genomic instability through telomeric DNA end fusions, resulting in mitotic catastrophe and apoptosis; and 4) induction of chromosomal instability by telomere aggregate formation.

Guanylate-binding protein 1 modulates proteasomal machinery in ovarian cancer.

Guanylate-binding protein 1 (GBP1) is known as an interferon-γ-induced GTPase. Here, we used genetically modified ovarian cancer (OC) cells to study the role of GBP1. The data generated show that GBP1 inhibition constrains the clonogenic potential of cancer cells. In vivo studies revealed that GBP1 overexpression in tumors promotes tumor progression and reduces median survival, whereas GBP1 inhibition delayed tumor progression with longer median survival. We employed proteomics-based thermal stability assay (CETSA) on GBP1 knockdown and overexpressed OC cells to study its molecular functions. CETSA results show that GBP1 interacts with many members of the proteasome. Furthermore, GBP1 inhibition sensitizes OC cells to paclitaxel treatment via accumulated ubiquitinylated proteins where GBP1 inhibition decreases the overall proteasomal activity. In contrast, GBP1-overexpressing cells acquired paclitaxel resistance via boosted cellular proteasomal activity. Overall, these studies expand the role of GBP1 in the activation of proteasomal machinery to acquire chemoresistance.

Augmenting Vaccine Efficacy against Delta Variant with 'Mycobacterium-w'-Mediated Modulation of NK-ADCC and TLR-MYD88 Pathways.

Mycobacterium-w (Mw) was shown to boost adaptive natural killer (ANK) cells and protect against COVID-19 during the first wave of the pandemic. As a follow-up of the trial, 50 healthcare workers (HCW) who had received Mw in September 2020 and subsequently received at least one dose of ChAdOx1 nCoV-19 vaccine (Mw + ChAdOx1 group) were monitored for symptomatic COVID-19 during a major outbreak with the delta variant of SARS-CoV-2 (April-June 2021), along with 201 HCW receiving both doses of the vaccine without Mw (ChAdOx1 group). Despite 48% having received just a single dose of the vaccine in the Mw + ChAdOx1 group, only two had mild COVID-19, compared to 36 infections in the ChAdOx1 group (HR-0.46, p = 0.009). Transcriptomic studies revealed an enhanced adaptive NK cell-dependent ADCC in the Mw + ChAdOx1 group, along with downregulation of the TLR2-MYD88 pathway and concomitant attenuation of downstream inflammatory pathways. This might have resulted in robust protection during the pandemic with the delta variant.

Methoxychalcone inhibitors of androgen receptor translocation and function.

Androgen receptor activity drives incurable castrate-resistant prostate cancer. All approved antiandrogens inhibit androgen receptor-driven transcription, and in addition the second-generation antiandrogen MDV3100 inhibits ligand-activated androgen receptor nuclear translocation, via an unknown mechanism. Here, we report methoxychalcones that lock the heat shock protein 90-androgen receptor complex in the cytoplasm in an androgen-non-responsive state, thus demonstrating a novel chemical scaffold for antiandrogen development and a unique mechanism of antiandrogen activity.

Arylalkyl ketones, benzophenones, desoxybenzoins and chalcones inhibit TNF-α induced expression of ICAM-1: structure-activity analysis.

The interaction between leukocytes and the vascular endothelial cells (EC) via cellular adhesion molecules plays an important role in the pathogenesis of various inflammatory and autoimmune diseases. Small molecules that block these interactions have been targeted as potential therapeutic agents against acute and chronic inflammatory diseases. In an effort to identify potent intercellular cell adhesion molecule-1 (ICAM-1) inhibitors, a large number of arylalkyl ketones, benzophenones, desoxybenzoins and chalcones and their analogs (54 in total) have been synthesized and screened for their ICAM-1 inhibitory activity. The structure-activity relationship studies of these compounds identified three potent chalcone derivatives and also demonstrated the possible mechanism for their ICAM-1 inhibitory activities. The most active compound was found to be 79.

Harnessing the predictive power of preclinical models for oncology drug development.

Recent progress in understanding the molecular basis of cellular processes, identification of promising therapeutic targets and evolution of the regulatory landscape makes this an exciting and unprecedented time to be in the field of oncology drug development. However, high costs, long development timelines and steep rates of attrition continue to afflict the drug development process. Lack of predictive preclinical models is considered one of the key reasons for the high rate of attrition in oncology. Generating meaningful and predictive results preclinically requires a firm grasp of the relevant biological questions and alignment of the model systems that mirror the patient context. In doing so, the ability to conduct both forward translation, the process of implementing basic research discoveries into practice, as well as reverse translation, the process of elucidating the mechanistic basis of clinical observations, greatly enhances our ability to develop effective anticancer treatments. In this Review, we outline issues in preclinical-to-clinical translatability of molecularly targeted cancer therapies, present concepts and examples of successful reverse translation, and highlight the need to better align tumour biology in patients with preclinical model systems including tracking of strengths and weaknesses of preclinical models throughout programme development.

A Cell-Based Screen Identifies HDAC Inhibitors as Activators of RIG-I Signaling.

Enhancing the immune microenvironment in cancer by targeting the nucleic acid sensors is becoming a potent therapeutic strategy. Among the nucleic acid sensors, activation of the RNA sensor Retinoic Acid-inducible Gene (RIG-I) using small hairpin RNAs has been shown to elicit powerful innate and adaptive immune responses. Given the challenges inherent in pharmacokinetics and delivery of RNA based agonists, we set out to discover small molecule agonists of RIG-I using a cell-based assay. To this end, we established and validated a robust high throughput screening assay based on a commercially available HEK293 reporter cell line with a luciferase reporter downstream of tandem interferon stimulated gene 54 (ISG54) promoter elements. We first confirmed that the luminescence in this cell line is dependent on RIG-I and the interferon receptor using a hairpin RNA RIG-I agonist. We established a 96-well and a 384-well format HTS based on this cell line and performed a proof-of-concept screen using an FDA approved drug library of 1,200 compounds. Surprisingly, we found two HDAC inhibitors Entinostat, Mocetinostat and the PLK1 inhibitor Volasertib significantly enhanced ISG-luciferase activity. This luminescence was substantially diminished in the null reporter cell line indicating the increase in signaling was dependent on RIG-I expression. Combination treatment of tumor cell lines with Entinostat increased RIG-I induced cell death in a mammary carcinoma cell line that is resistant to either Entinostat or RIG-I agonist alone. Taken together, our data indicates an unexpected role for HDAC1,-3 inhibitors in enhancing RIG-I signaling and highlight potential opportunities for therapeutic combinations.

Designing Novel BCR-ABL Inhibitors for Chronic Myeloid Leukemia with Improved Cardiac Safety.

Development of tyrosine kinase inhibitors (TKIs) targeting the BCR-ABL oncogene constitutes an effective approach for the treatment of chronic myeloid leukemia (CML) and/or acute lymphoblastic leukemia. However, currently available inhibitors are limited by drug resistance and toxicity. Ponatinib, a third-generation inhibitor, has demonstrated excellent efficacy against both wild type and mutant BCR-ABL kinase, including the "gatekeeper" T315I mutation that is resistant to all other currently available TKIs. However, it is one of the most cardiotoxic of the FDA-approved TKIs. Herein, we report the structure-guided design of a novel series of potent BCR-ABL inhibitors, particularly for the T315I mutation. Our drug design paradigm was coupled to iPSC-cardiomyocyte models. Systematic structure-activity relationship studies identified two compounds, 33a and 36a, that significantly inhibit the kinase activity of both native BCR-ABL and the T315I mutant. We have identified the most cardiac-safe TKIs reported to date, and they may be used to effectively treat CML patients with the T315I mutation.

A New Nrf2 Inhibitor Enhances Chemotherapeutic Effects in Glioblastoma Cells Carrying p53 Mutations.

TP53 tumor suppressor gene is a commonly mutated gene in cancer. p53 mediated senescence is critical in preventing oncogenesis in normal cells. Since p53 is a transcription factor, mutations in its DNA binding domain result in the functional loss of p53-mediated cellular pathways. Similarly, nuclear factor erythroid 2-related factor 2 (Nrf2) is another transcription factor that maintains cellular homeostasis by regulating redox and detoxification mechanisms. In glioblastoma (GBM), Nrf2-mediated antioxidant activity is upregulated while p53-mediated senescence is lost, both rendering GBM cells resistant to treatment. To address this, we identified novel Nrf2 inhibitors from bioactive compounds using a molecular imaging biosensor-based screening approach. We further evaluated the identified compounds for their in vitro and in vivo chemotherapy enhancement capabilities in GBM cells carrying different p53 mutations. We thus identified an Nrf2 inhibitor that is effective in GBM cells carrying the p53 (R175H) mutation, a frequent clinically observed hotspot structural mutation responsible for chemotherapeutic resistance in GBM. Combining this drug with low-dose chemotherapies can potentially reduce their toxicity and increase their efficacy by transiently suppressing Nrf2-mediated detoxification function in GBM cells carrying this important p53 missense mutation.

Impact of an Immune Modulator Mycobacterium-w on Adaptive Natural Killer Cells and Protection Against COVID-19.

The kinetics of NKG2C+ adaptive natural killer (ANK) cells and NKG2A+inhibitory NK (iNK) cells with respect to the incidence of SARS-CoV-2 infection were studied for 6 months in a cohort of healthcare workers following the administration of the heat-killed Mycobacterium w (Mw group) in comparison to a control group. In both groups, corona virus disease 2019 (COVID-19) correlated with lower NKG2C+ANK cells at baseline. There was a significant upregulation of NKG2C expression and IFN-γ release in the Mw group (p=0.0009), particularly in those with a lower baseline NKG2C expression, along with the downregulation of iNK cells (p<0.0001). This translated to a significant reduction in the incidence and severity of COVID-19 in the Mw group (incidence risk ratio-0.15, p=0.0004). RNA-seq analysis at 6 months showed an upregulation of the ANK pathway genes and an enhanced ANK-mediated antibody-dependent cellular cytotoxicity (ADCC) signature. Thus, Mw was observed to have a salutary impact on the ANK cell profile and a long-term upregulation of ANK-ADCC pathways, which could have provided protection against COVID-19 in a non-immune high-risk population.