Mechanism-based Screening Research Articles

Mechanism-based toxicity screening of organophosphate flame retardants using Tox21 assays and molecular docking analysis

As brominated flame retardants are phased out and regulations on their use become stricter, concerns over organophosphate flame retardants (OPFRs) have increased due to their high production. In response, this study aimed to screen the potential toxicity of emerging OPFRs using in vitro Tox21 assays and in silico molecular docking analysis. For 48 OPFRs collected from the literature, we investigated their bioactivity with human nuclear receptors using Tox21 data, focusing on pathways related to endocrine disruption (ERs, AR), stress response (GR), energy homeostasis (PPARs, FXR), and detoxification (PXR, CAR). For OPFRs not tested in Tox21 assays, molecular docking simulations were performed to predict binding potential. Results showed that CAR/PXR and FXR had relatively high reactivity with diverse OPFRs, indicating potential molecular initiating events (MIEs). Among the 48 OPFRs, 28 interacted with one or more receptors, suggesting they may act as potential stressors of adverse outcome pathways (AOPs) leading to various human diseases. Aryl- and halogenated-OPFRs displayed higher bioactivity compared to alkyl-OPFRs. Additionally, as the logKow value and carbon number of OPFRs increased, their interaction with nuclear receptors also increased. These structure- and physicochemistry-dependent bioactivities provide insights for designing safer OPFRs to avoid regrettable substitutions. Of these prioritized OPFRs, 13 showed low oral points-of-departure (POD) values under 100 mg/kg/day. In contrast, the other 15 OPFRs lacked sufficient data or exhibited less severe toxicity, despite being predicted to be of high concern in our analysis. Since several OPFRs are commonly used in consumer products that can lead to daily human exposure, we suggest that these OPFRs have the potential to reveal undisclosed effects and should therefore undergo further assessment.

Molecular mechanism-based extractant screening and process design for the separation of methanol/dimethyl carbonate azeotrope

The separation of methanol/dimethyl carbonate azeotrope is an important process in the industrial production of dimethyl carbonate. Extractive distillation is an effective separation method, which can ensure the full utilization of resources and achieve sustainable development. In this work, an extractant screening method based on density functional theory (DFT) was proposed. By comparing the interaction energy difference, the optimal extractant (dimethyl sulfoxide, DMSO) was obtained. The molecular mechanism of action between the extractant and the separated components was explained. The separation order of the components of the extraction distillation process was obtained by the magnitude of the interaction energy. The σ-profile curve and isovolatility curves method were used to verify the effectiveness of the proposed method. A new separation process with DMSO as an extractant was designed. With the lowest total annual cost (TAC) as the goal, sensitivity analysis and cyclic iteration method is used to optimize the parameters involved in the process to determine the best process parameters. Heat integration technology is introduced to further reduce energy consumption. TAC, energy utilization efficiency, and global warming potential (GWP) were used to evaluate the process. Compared with the reported optimal extractant methyl salicylate, the TAC of the simple process using DMSO as the extractant was reduced by 9.08%. The heat-integrated process with DMSO shows a TAC reduction of 17.17% compared to the simple process. And the energy utilization efficiency and GWP were increased by 40.90% and decreased by 31.88%, respectively. The results show that the extractant screened by DFT method is applied to the azeotrope separation, which makes the process have good economic benefits, low energy consumption, and less environmental impact.

Oxidative adsorption mechanism-based screening of zeolites for deep purification and recycling of NOx from humid gases

Adsorption is a promising technology for deep purification of nitrogen oxides (NOx) from flue gases and simultaneously recycling NO2 as an economically valuable chemical. Zeolite as a robust NOx adsorbent provides the key step for success in practical applications, of which the screening remains challenging regarding the NO-NO2 oxidation and adsorption combined process. In this work, experimental evaluations on practical NOx sorption performances on series of zeolites were conducted. Effects of the zeolite typological structure, compensating cation, and SiO2/Al2O3 ratio on deep purification efficacy, water resistance, and regenerability at varying humidity were obtained, revealing the importance of kinetics of the NO oxidation and NO2 physisorption consecutive process. The H-typed 10-membered-ring straight-channel zeolites (MFI and FER) were demonstrated to be the preferred NOx adsorbents qualified for industrial needs, as compared to other caged-channel, hybrid-channel, larger-sized straight-channel, or Na-typed counterparts. Deeper insights into the key role of Brønsted acid sites (BAS) in driving the NO-NO2 catalytic cycle and promoting the adsorption competitiveness of NOx over water vapor were revealed based on in-situ diffuse reflectance Fourier transform infrared spectroscopy characterizations. The MFI zeolite with improved performance at the low-silica version could benefit from the efficient utilization of BAS within the highly accessible medium-sized straight channel, while the FAU zeolite (representative caged-channel zeolite) requiring appropriate SiO2/Al2O3 ratio for optimal BAS utilization may suffer from the limited site accessibility. The findings clarify the relationships between NOx oxidative adsorption performances and zeolite properties, which provides a fundamental perspective and scientific screening strategy for NOx adsorbents.

Emerging of Three-Dimensional Models To Study Human Diseases

For several decades, biomedical research has traditionally relied on the use of two-dimensional (2D) cell culture and animal models to study human biology and diseases. However, it can be difficult to address questions that are relevant to human biology and diseases in animal models or 2D cell culture, such as genetics, development, immune response, and cell host tropism. Furthermore, scarcity of advanced in vitro and in vivo models has hindered progress in understanding disease causes and, as a result, the creation of innovative therapeutic methods for patients. To improve our understanding and the current situation, more physiologically and realistic research models for mechanism-based target identification, drug development and screening, developmental biology, cancer and infectious diseases are needed.

Fast identification of off-target liabilities in early antibiotic discovery with Fourier-transform infrared spectroscopy.

Structural modifications of known antibiotic scaffolds have kept the upper hand on resistance, but we are on the verge of not having antibiotics for many common infections. Mechanism-based discovery assays reveal novelty, exclude off-target liabilities, and guide lead optimization. For that, we developed a fast and automatable protocol using high-throughput Fourier-transform infrared spectroscopy (FTIRS). Metabolic fingerprints of Staphylococcus aureus and Escherichia coli exposed to 35 compounds, dissolved in dimethyl sulfoxide (DMSO) or water, were acquired. Our data analysis pipeline identified biomarkers of off-target effects, optimized spectral preprocessing, and identified the top-performing machine learning algorithms for off-target liabilities and mechanism of action (MOA) identification. Spectral bands with known biochemical associations more often yielded more significant biomarkers of off-target liabilities when bacteria were exposed to compounds dissolved in water than DMSO. Highly discriminative models distinguished compounds with predominant off-target effects from antibiotics with well-defined MOA (AUROC > 0.87, AUPR > 0.79, F1 > 0.81), and from the latter predicted their MOA (AUROC > 0.88, AUPR > 0.70, F1 > 0.70). The compound solvent did not affect predictive models. FTIRS is fast, simple, inexpensive, automatable, and highly effective at predicting MOA and off-target liabilities. As such, FTIRS mechanism-based screening assays can be applied for hit discovery and to guide lead optimization during the early stages of antibiotic discovery.

Abstract 1259: Preclinical characterization of LY3537982, a novel, highly selective and potent KRAS-G12C inhibitor

Abstract KRAS-G12C is an important oncogenic mutation in patients with NSCLC, CRC, and other cancer types. Currently, there are no FDA-approved KRAS-G12C inhibitors, and those in clinical development have relatively modest activity compared to other approved therapies targeting other classic oncogenic drivers. This modest activity may be potentially due in part to incomplete target occupancy and trapping of mutant KRAS in the inactive GDP-bound state. Achieving maximal clinical benefit in patients harboring a KRAS-G12C mutation, may require a potent inhibitor capable of achieving near complete target engagement. Here, we report the identification of LY3537982, a novel, highly selective and potent inhibitor of the KRAS-G12C protein, discovered using structure-based design. In kinetic studies, LY3537982 showed a high Kinact/Ki value (248,016 M-1 s-1), compared to AMG510 (7,220 M-1 s-1) and MRTX849 (35,000 M-1 s-1). LY3537982 inhibited KRAS-GTP loading with an IC50 value of 3.35 nM in the KRAS-G12C mutant H358 lung cancer cell line, while AMG510 and MRTX849 had IC50 values of 47.9 nM and 89.9 nM, respectively. LY3537982 also inhibited phospho-ERK in H358 cells with an IC50 value of 0.65 nM, while the IC50 values of AMG510 and MRTX849 were 13.5 nM and 14 nM, respectively. In a panel of cancer cell lines with KRAS-G12C or non-G12C mutations, LY3537982 selectively inhibited the growth of KRAS-G12C mutant tumor cells and not KRAS wild-type or non-G12C mutant cells. Sensitivity to LY3537982 varied among the KRAS-G12C mutant cells tested, suggesting that not all cell lines maintain the same dependence on KRAS-G12C. Similarly, in multiple xenograft or patient-derived xenograft (PDX) models harboring a KRAS-G12C mutation, LY3537982 exhibited a range of anti-tumor activity from complete regression to significant tumor growth inhibition, at 3 to 30 mg/kg QD or BID. Mechanism-based combinational screens have also identified certain targeted therapies that can synergize with LY3537982 to achieve better anti-tumor activity in vitro and in vivo, including abemaciclib, the selective AurA inhibitor LY3295668, and cetuximab. Together these data suggest that in certain biologic contexts, broader and more durable anti-tumor activity could be achieved with combination regimens. A first-in-human Phase 1 clinical trial is planned for 2021. Citation Format: Sheng-Bin Peng, Chong Si, Youyan Zhang, Robert D. Van Horn, Xi Lin, Xueqian Gong, Lysiane Huber, Gregory Donoho, Carmen Curtis, John M. Strelow, Wayne P. Bocchinfuso, Deqi Guo, Serge L. Boulet, David Barda, Danalyn Manglicmot, Melbert-Brian D. Saflor, Jing Wang, Junpeng Xiao, Michael J. Chalmers, Lee Burns, Ryan J. Linder, Bradley L. Ackermann, Paul D. Cornwell, Lian Zhou, Denis McCann, James Henry. Preclinical characterization of LY3537982, a novel, highly selective and potent KRAS-G12C inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1259.

D2O-Probed Raman Microspectroscopy Distinguishes the Metabolic Dynamics of Macromolecules in Organellar Anticancer Drug Response.

To profile the metabolic dynamics responding to drugs at the single-cell/organelle resolution, rapid and economical mechanism-revealing methods are required. Here, we introduced D2O-probed Raman microspectroscopy in combination with the multivariate curve resolution-alternating least squares (MCR-ALS or MCR) algorithm. Exploiting MCR to deconvolute each macromolecular component specifically, the method is able to track and distinguish changes in lipid and protein metabolic activities in a human cancer cell line (MCF-7) and in Saccharomyces cerevisiae, in response to the metabolism-inhibitory effect of rapamycin, which inhibits the mammalian/mechanistic target of rapamycin (mTOR) signaling. Under rapamycin, in the lipid bodies of cancer cells, metabolic activities of both protein and lipid are suppressed; in the nucleus, protein synthesis remains active, whereas lipid synthesis is inhibited; in the cytoplasm, syntheses of protein and lipid are both dose- and duration-dependent. Thus, rapamycin differentially influences protein and lipid synthesis in mTOR signaling. Moreover, the strong correlation between macromolecular-specific components of yeast and those in MCF-7 cytoplasm, nucleus, and lipid bodies revealed similarity in rapamycin response. Notably, highly metabolically active cancer cells after high-dosage rapamycin exposure (500 or 5000 × IC50) were revealed, which escape detection by population-level cytotoxicity tests. Thus, by unveiling macromolecule-specific metabolic dynamics at the organelle level, the method is valuable to mechanism-based rapid screening and dissection of drug response.

Employing Genetically Encoded, Biophysical Sensors to Understand WNT/Frizzled Interaction and Receptor Complex Activation.

The Frizzled (FZD) family of WNT receptors consists of ten paralogues in mammals. They belong to the superfamily of G protein-coupled receptors and regulate crucial processes during embryonic development. Dysregulated FZD signaling leads to disease, most prominently to diverse forms of cancer, which renders these receptors attractive for drug discovery. Recent advances in assay development and the design of genetically encoded biosensors monitoring ligand-receptor interaction, conformational dynamics, and protein-protein interaction have allowed for a better pharmacological understanding of WNT/FZD signal transduction and open novel avenues for mechanism-based drug discovery and screening. In this chapter, we summarize the recent progress in the molecular dissection of FZD activation based on advanced biosensors.

Facial fractures and associated injuries in high- versus low-energy trauma: all are not created equal

IntroductionFacial fractures (FFs) occur after high- and low-energy trauma; differences in associated injuries and outcomes have not been well articulated.ObjectiveTo compare the epidemiology, management, and outcomes of patients suffering FFs from high-energy and low-energy mechanisms.MethodsWe conducted a 6-year retrospective local trauma registry analysis of adults aged 18–55 years old that suffered a FF treated at the Santa Barbara Cottage Hospital. Fracture patterns, concomitant injuries, procedures, and outcomes were compared between patients that suffered a high-energy mechanism (HEM: motor vehicle crash, bicycle crash, auto versus pedestrian, falls from height > 20 feet) and those that suffered a low-energy mechanism (LEM: assault, ground-level falls) of injury.ResultsFFs occurred in 123 patients, 25 from an HEM and 98 from an LEM. Rates of Le Fort (HEM 12% vs. LEM 3%, P = 0.10), mandible (HEM 20% vs. LEM 38%, P = 0.11), midface (HEM 84% vs. LEM 67%, P = 0.14), and upper face (HEM 24% vs. LEM 13%, P = 0.217) fractures did not significantly differ between the HEM and LEM groups, nor did facial operative rates (HEM 28% vs. LEM 40%, P = 0.36). FFs after an HEM event were associated with increased Injury Severity Scores (HEM 16.8 vs. LEM 7.5, P <0.001), ICU admittance (HEM 60% vs. LEM 13.3%, P <0.001), intracranial hemorrhage (ICH) (HEM 52% vs. LEM 15%, P <0.001), cervical spine fractures (HEM 12% vs. LEM 0%, P = 0.008), truncal/lower extremity injuries (HEM 60% vs. LEM 6%, P <0.001), neurosurgical procedures for the management of ICH (HEM 54% vs. LEM 36%, P = 0.003), and decreased Glasgow Coma Score on arrival (HEM 11.7 vs. LEM 14.2, P <0.001).ConclusionFFs after HEM events were associated with severe and multifocal injuries. FFs after LEM events were associated with ICH, concussions, and cervical spine fractures. Mechanism-based screening strategies will allow for the appropriate detection and management of injuries that occur concomitant to FFs.Type of studyRetrospective cohort study.Level of evidenceLevel III.

The Current Understanding of Autophagy in Nanomaterial Toxicity and Its Implementation in Safety Assessment-Related Alternative Testing Strategies.

Nanotechnology has rapidly promoted the development of a new generation of industrial and commercial products; however, it has also raised some concerns about human health and safety. To evaluate the toxicity of the great diversity of nanomaterials (NMs) in the traditional manner, a tremendous number of safety assessments and a very large number of animals would be required. For this reason, it is necessary to consider the use of alternative testing strategies or methods that reduce, refine, or replace (3Rs) the use of animals for assessing the toxicity of NMs. Autophagy is considered an early indicator of NM interactions with cells and has been recently recognized as an important form of cell death in nanoparticle-induced toxicity. Impairment of autophagy is related to the accelerated pathogenesis of diseases. By using mechanism-based high-throughput screening in vitro, we can predict the NMs that may lead to the generation of disease outcomes in vivo. Thus, a tiered testing strategy is suggested that includes a set of standardized assays in relevant human cell lines followed by critical validation studies carried out in animals or whole organism models such as C. elegans (Caenorhabditis elegans), zebrafish (Danio rerio), and Drosophila (Drosophila melanogaster)for improved screening of NM safety. A thorough understanding of the mechanisms by which NMs perturb biological systems, including autophagy induction, is critical for a more comprehensive elucidation of nanotoxicity. A more profound understanding of toxicity mechanisms will also facilitate the development of prevention and intervention policies against adverse outcomes induced by NMs. The development of a tiered testing strategy for NM hazard assessment not only promotes a more widespread adoption of non-rodent or 3R principles but also makes nanotoxicology testing more ethical, relevant, and cost- and time-efficient.

ToxTracker Reporter Cell Lines as a Tool for Mechanism-Based (Geno)Toxicity Screening of Nanoparticles—Metals, Oxides and Quantum Dots

The increased use of nanoparticles (NPs) requires efficient testing of their potential toxic effects. A promising approach is to use reporter cell lines to quickly assess the activation of cellular stress response pathways. This study aimed to use the ToxTracker reporter cell lines to investigate (geno)toxicity of various metal- or metal oxide NPs and draw general conclusions on NP-induced effects, in combination with our previous findings. The NPs tested in this study (n = 18) also included quantum dots (QDs) in different sizes. The results showed a large variation in cytotoxicity of the NPs tested. Furthermore, whereas many induced oxidative stress only few activated reporters related to DNA damage. NPs of manganese (Mn and Mn3O4) induced the most remarkable ToxTracker response with activation of reporters for oxidative stress, DNA damage, protein unfolding and p53-related stress. The QDs (CdTe) were highly toxic showing clearly size-dependent effects and calculations suggest surface area as the most relevant dose metric. Of all NPs investigated in this and previous studies the following induce the DNA damage reporter; CuO, Co, CoO, CdTe QDs, Mn, Mn3O4, V2O5, and welding NPs. We suggest that these NPs are of particular concern when considering genotoxicity induced by metal- and metal oxide NPs.

MRGPRX2 activation as a rapid, high-throughput mechanistic-based approach for detecting peptide-mediated human mast cell degranulation liabilities

Mast cells play key roles in allergy, anaphylaxis/anaphylactoid reactions, and defense against pathogens/toxins. These cells contain cytoplasmic granules with a wide spectrum of pleotropic mediators that are released upon activation. While mast cell degranulation (MCD) occurs upon clustering of the IgE receptor bound to IgE and antigen, MCD is also triggered through non-IgE-mediated mechanisms, one of which is via Mas-related G protein-coupled receptor X2 (MRGPRX2). MRGPRX2 can be activated by many basic biogenic amines and peptides. Consequently, MRGPRX2-mediated MCD is an important potential safety liability for peptide therapeutics. To facilitate peptide screening for this liability in early preclinical drug development, a rapid, high-throughput engineered CHO-K1 cell-based MRGPRX2 activation assay was evaluated and compared to histamine release in CD34+ stem cell-derived mature human mast cells as a reference assay, using 30 positive control and 29 negative control peptides for MCD. Both G protein-dependent (Ca2+ endpoint) and -independent (β-arrestin endpoint) pathways were assessed in the MRGPRX2 activation assay. The MRGPRX2 activation assay had a sensitivity of 100% for both Ca2+ and β-arrestin endpoints and a specificity of 93% (β-arrestin endpoint) and 83% (Ca2+ endpoint) compared to histamine release in CD34+ stem cell-derived mature human mast cells. These findings suggest that assessing MRGPRX2 activation in an engineered cell model can provide value as a rapid, high-throughput, economical mechanism-based screening tool for early MCD hazard identification during preclinical safety evaluation of peptide-based therapeutics.

Relationship of Pain Quality Descriptors and Quantitative Sensory Testing: Sickle Cell Disease.

Chronic pain in adults with sickle cell disease (SCD) may be the result of altered processing in the central nervous system, as indicated by quantitative sensory testing (QST). Sensory pain quality descriptors on the McGill Pain Questionnaire (MPQ) are indicators of typical or altered pain mechanisms but have not been validated with QST-derived classifications. The specific aim of this study was to identify the sensory pain quality descriptors that are associated with the QST-derived normal or sensitized classifications. We expected to find that sets of sensory pain quality descriptors would discriminate the classifications. A cross-sectional quantitative study of existing data from 186 adults of African ancestry with SCD. Variables included MPQ descriptors, patient demographic data, and QST-derived classifications. The participants were classified as central sensitization (n = 33), mixed sensitization (n = 23), and normal sensation. Sensory pain quality descriptors that differed statistically between mixed sensitization and central sensation compared to normal sensitization included cold (p = .01) and spreading (p = .01). Aching (p = .01) and throbbing (p = .01) differed statistically between central sensitization compared with mixed sensitization and normal sensation. Beating (p = .01) differed statistically between mixed sensitization compared with central sensitization and normal sensation. No set of sensory pain quality descriptors differed statistically between QST classifications. Our study is the first to examine the association between MPQ sensory pain quality descriptors and QST-derived classifications in adults with SCD. Our findings provide the basis for the development of a MPQ subscale with potential as a mechanism-based screening tool for neuropathic pain.

The mechanism-based toxicity screening of particles with use in the food and nutrition sector via the ToxTracker reporter system

The rapid expansion of the incorporation of nano-sized materials in consumer products overlaps with the necessity for high-throughput reliable screening tools for the identification of the potential hazardous properties of the nanomaterials. The ToxTracker assay (mechanism-based reporter assay based on embryonic stem cells that uses GFP-tagged biomarkers for detection of DNA damage, oxidative stress and general cellular stress) is one such tool, which could prove useful in the field of particle toxicology allowing for high throughput screening. Here, ToxTracker was utilised to evaluate the potential hazardous properties of two particulates currently used in the food industry (vegetable carbon (E153) and food-grade TiO2 (E171)). Due to the fact that ToxTracker is based on a stem cell format, it is crucial that the data generated is assessed for its suitability and comparability to more conventionally used relevant source of cells - in this case cells from the gastrointestinal tract and the liver. Therefore, the cell reporter findings were compared to data from traditional assays (cytotoxicity, anti-oxidant depletion and DNA damage) and tissue relevant cell types. The data showed E171 to be the most cytotoxic, decreased intracellular glutathione and the most significant with regards to genotoxic effects. The ToxTracker data showed comparability to conventional toxicity and oxidative stress assays; however, some discrepancies were evident between the findings from ToxTracker and the comet assay.

Lens cholesterol biosynthesis inhibition: A common mechanism of cataract formation in laboratory animals by pharmaceutical products.

CJ-12,918, a 5-lipoxygenase (5-LO) inhibitor, caused cataracts during a 1-month safety assessment studies in rats whereas the structurally similar ZD-2138 was without effect. For CJ-12,918 analogs, blocking different sites of metabolic liability reduced (CJ-13,454) and eliminated (CJ-13,610) cataract formation in both rats and dogs. Using this chemical series as a test set, models and mechanisms of toxicity were first explored by testing the utility of ex vivo rat lens explant cultures as a safety screen. This model overpredicted the cataractogenic potential of ZD-2138 due to appreciably high lens drug levels and was abandoned in favor of a mechanism-based screen. Perturbations in lens sterol content, from a decline in lathosterol content, preceded cataract formation suggesting CJ-12,918 inhibited lens cholesterol biosynthesis (LCB). A 2-day bioassay in rats using ex vivo LCB assessments showed that the level of LCB inhibition was correlated with incidence of cataract formation in animal studies by these 5-LO inhibitors. Thereafter, this 2-day bioassay was applied to other pharmaceutical programs (neuronal nitric oxide synthase, sorbitol dehydrogenase inhibitor, squalene synthetase inhibitor and stearoyl-CoA desaturase-1 inhibitors/D4 antagonists) that demonstrated cataract formation in either rats or dogs. LCB inhibition >40% was associated with a high incidence of cataract formation in both rats and dogs that was species specific. Bioassay sensitivity/specificity were further explored with positive (RGH-6201/ciglitazone/U18666A) and negative (tamoxifen/naphthalene/galactose) mechanistic controls. This body of work over two decades shows that LCB inhibition was a common mechanism of cataract formation by pharmaceutical agents and defined a level of inhibition >40% that was typically associated with causing cataracts in safety assessment studies typically ≥1month.

High-throughput screens identify HSP90 inhibitors as potent therapeutics that target inter-related growth and survival pathways in advanced prostate cancer

The development of new treatments for castrate resistant prostate cancer (CRPC) must address such challenges as intrinsic tumor heterogeneity and phenotypic plasticity. Combined PTEN/TP53 alterations represent a major genotype of CRPC (25–30%) and are associated with poor outcomes. Using tumor-derived, castration-resistant Pten/Tp53 null luminal prostate cells for comprehensive, high-throughput, mechanism-based screening, we identified several vulnerabilities among >1900 compounds, including inhibitors of: PI3K/AKT/mTOR, the proteasome, the cell cycle, heat shock proteins, DNA repair, NFκB, MAPK, and epigenetic modifiers. HSP90 inhibitors were one of the most active compound classes in the screen and have clinical potential for use in drug combinations to enhance efficacy and delay the development of resistance. To inform future design of rational drug combinations, we tested ganetespib, a potent second-generation HSP90 inhibitor, as a single agent in multiple CRPC genotypes and phenotypes. Ganetespib decreased growth of endogenous Pten/Tp53 null tumors, confirming therapeutic activity in situ. Fifteen human CRPC LuCaP PDX-derived organoid models were assayed for responses to 110 drugs, and HSP90 inhibitors (ganetespib and onalespib) were among the select group of drugs (<10%) that demonstrated broad activity (>75% of models) at high potency (IC50 <1 µM). Ganetespib inhibits multiple targets, including AR and PI3K pathways, which regulate mutually compensatory growth and survival signals in some forms of CRPC. Combined with castration, ganetespib displayed deeper PDX tumor regressions and delayed castration resistance relative to either monotherapy. In all, comprehensive data from near-patient models presents novel contexts for HSP90 inhibition in multiple CRPC genotypes and phenotypes, expands upon HSP90 inhibitors as simultaneous inhibitors of oncogenic signaling and resistance mechanisms, and suggests utility for combined HSP90/AR inhibition in CRPC.

Integrating mechanism-based screening paradigm into homology and de novo modeling exemplified by Mycobacterium Tuberculosis 30S ribosomal structure and its potential application as a screening target

Integrating mechanism-based screening paradigm into homology and de novo modeling exemplified by Mycobacterium Tuberculosis 30S ribosomal structure and its potential application as a screening target

A covalent PIN1 inhibitor selectively targets cancer cells by a dual mechanism of action

The prolyl isomerase PIN1, a critical modifier of multiple signalling pathways, is overexpressed in the majority of cancers and its activity strongly contributes to tumour initiation and progression. Inactivation of PIN1 function conversely curbs tumour growth and cancer stem cell expansion, restores chemosensitivity and blocks metastatic spread, thus providing the rationale for a therapeutic strategy based on PIN1 inhibition. Notwithstanding, potent PIN1 inhibitors are still missing from the arsenal of anti-cancer drugs. By a mechanism-based screening, we have identified a novel covalent PIN1 inhibitor, KPT-6566, able to selectively inhibit PIN1 and target it for degradation. We demonstrate that KPT-6566 covalently binds to the catalytic site of PIN1. This interaction results in the release of a quinone-mimicking drug that generates reactive oxygen species and DNA damage, inducing cell death specifically in cancer cells. Accordingly, KPT-6566 treatment impairs PIN1-dependent cancer phenotypes in vitro and growth of lung metastasis in vivo.

Regioselective Synthesis of 2-Substituted Indoles from Benzotriazoles and Alkynes by Photoinitiated Denitrogenation

Herein, we describe a photoinitiated and regioselective synthesis of 2-substituted indoles under mild reaction conditions. This biologically privileged scaffold was accessed in good yields from N-aroylbenzotriazoles, a quencher class previously identified using our mechanism-based luminescence screening, and terminal alkynes in the presence of a photocatalyst and blue light irradiation. This straightforward protocol displays a broad substrate scope and functional group tolerance. Furthermore, the mildness and robustness of the reaction were assessed by the application of an additive-based robustness screen. The determination of the reaction quantum yield and Stern–Volmer studies support the proposed photoinitiated radical chain mechanism.

Diverse Visible-Light-Promoted Functionalizations of Benzotriazoles Inspired by Mechanism-Based Luminescence Screening.

Three new visible-light-promoted functionalizations of benzotriazole substrates were discovered using a mechanism-based screening method. ortho-Thiolated, borylated, and alkylated N-arylbenzamide products were obtained under mild reaction conditions in a new denitrogenative synthetic approach to functionalized aniline derivatives. The functional group tolerance of the borylation reaction was further analyzed in the first application of an additive-based robustness screen in a photocatalytic transformation. All the functionalizations proceed via photocatalytically initiated chain mechanisms as indicated by determination of the reaction quantum yields and Stern-Volmer analyses.