Replacement of the hydroxamic acid group in the selective HDAC8 inhibitor PCI-34051.

PCI-34051 is a valuable tool to interrogate the therapeutic effects of selective inhibition of HDAC8. However, it has not advanced to clinical trials, perhaps due to poor PK or off-target effects. We hypothesized that the presence of a hydroxamic acid (HA) group in PCI-34051 contributed to its lack of advancement. Therefore, we replaced the HA in the PCI-34051 scaffold with a series of moieties that have the potential to bind to Zn and evaluated their activity in a HDAC8 assay. Surprisingly, none of the replacements effectively mimicked the HA, and analogs lost significant potency. Evaluation of the analogs' affinity to Zn indicated that none had affinity for Zn within the same range as the HA. These studies point to the difficulty in the application of bioisosteric replacements for Zn binding motifs.

Characterization of allosteric modulators that disrupt androgen receptor co-activator protein-protein interactions to alter transactivation-Drug leads for metastatic castration resistant prostate cancer.

Three series of compounds were prioritized from a high content screening campaign that identified molecules that blocked dihydrotestosterone (DHT) induced formation of Androgen Receptor (AR) protein-protein interactions (PPIs) with the Transcriptional Intermediary Factor 2 (TIF2) coactivator and also disrupted preformed AR-TIF2 PPI complexes; the hydrobenzo-oxazepins (S1), thiadiazol-5-piperidine-carboxamides (S2), and phenyl-methyl-indoles (S3). Compounds from these series inhibited AR PPIs with TIF2 and SRC-1, another p160 coactivator, in mammalian 2-hybrid assays and blocked transcriptional activation in reporter assays driven by full length AR or AR-V7 splice variants. Compounds inhibited the growth of five prostate cancer cell lines, with many exhibiting differential cytotoxicity towards AR positive cell lines. Representative compounds from the 3 series substantially reduced both endogenous and DHT-enhanced expression and secretion of the prostate specific antigen (PSA) cancer biomarker in the C4-2 castration resistant prostate cancer (CRPC) cell line. The comparatively weak activities of series compounds in the H3-DHT and/or TIF2 box 3 LXXLL-peptide binding assays to the recombinant ligand binding domain of AR suggest that direct antagonism at the orthosteric ligand binding site or AF-2 surface respectively are unlikely mechanisms of action. Cellular enhanced thermal stability assays (CETSA) indicated that compounds engaged AR and reduced the maximum efficacy and right shifted the EC50 of DHT-enhanced AR thermal stabilization consistent with the effects of negative allosteric modulators. Molecular docking of potent representative hits from each series to AR structures suggest that S1-1 and S2-6 engage a novel binding pocket (BP-1) adjacent to the orthosteric ligand binding site, while S3-11 occupies the AR binding function 3 (BF-3) allosteric pocket. Hit binding poses indicate spaces and residues adjacent to the BP-1 and BF-3 pockets that will be exploited in future medicinal chemistry optimization studies. Small molecule allosteric modulators that prevent/disrupt AR PPIs with coactivators like TIF2 to alter transcriptional activation in the presence of orthosteric agonists might evade the resistance mechanisms to existing prostate cancer drugs and provide novel starting points for medicinal chemistry lead optimization and future development into therapies for metastatic CRPC.

Validation of HDAC8 Inhibitors as Drug Discovery Starting Points to Treat Acute Kidney Injury.

Acute kidney injury (AKI), a sudden loss of kidney function, is a common and serious condition for which there are no approved specific therapies. While there are multiple approaches to treat the underlying causes of AKI, no targets have been clinically validated. Here, we assessed a series of potent, selective competitive inhibitors of histone deacetylase 8 (HDAC8), a promising therapeutic target in an AKI setting. Using biochemical assays, zebrafish AKI phenotypic assays, and human kidney organoid assays, we show that selective HDAC8 inhibitors can lead to efficacy in increasingly stringent models. One of these, PCI-34051, was efficacious in a rodent model of AKI, further supporting the potential for HDAC8 inhibitors and, in particular, this scaffold as a therapeutic approach to AKI.

Detection and impact of hypoxic regions in multicellular tumor spheroid cultures formed by head and neck squamous cell carcinoma cells lines.

In solid tumors like head and neck cancer (HNC), chronic and acute hypoxia have serious adverse clinical consequences including poorer overall patient prognosis, enhanced metastasis, increased genomic instability, and resistance to radiation-, chemo-, or immuno-therapies. However, cells in the two-dimensional monolayer cultures typically used for cancer drug discovery experience 20%-21% O2 levels (normoxic) which are 4-fold higher than O2 levels in normal tissues and ≥10-fold higher than in the hypoxic regions of solid tumors. The oxygen electrodes, exogenous bio-reductive markers, and increased expression of endogenous hypoxia-regulated proteins like HIF-1α generally used to mark hypoxic regions in solid tumors are impractical in large sample numbers and longitudinal studies. We used a novel homogeneous live-cell permeant HypoxiTRAK™ (HPTK) molecular probe compatible with high content imaging detection, analysis, and throughput to identify and quantify hypoxia levels in live HNC multicellular tumor spheroid (MCTS) cultures over time. Accumulation of fluorescence HPTK metabolite in live normoxic HNC MCTS cultures correlated with hypoxia detection by both pimonidazole and HIF-1α staining. In HNC MCTSs, hypoxic cytotoxicity ratios for the hypoxia activated prodrugs (HAP) evofosfamide and tirapazamine were much smaller than have been reported for uniformly hypoxic 2D monolayers in gas chambers, and many viable cells remained after HAP exposure. Cells in solid tumors and MCTSs experience three distinct O2 microenvironments dictated by their distances from blood vessels or MCTS surfaces, respectively; oxic, hypoxic, or intermediate levels of hypoxia. These studies support the application of more physiologically relevant in vitro 3D models that recapitulate the heterogeneous microenvironments of solid tumors for preclinical cancer drug discovery.

Unbiased High-Throughput Drug Combination Pilot Screening Identifies Synergistic Drug Combinations Effective against Patient-Derived and Drug-Resistant Melanoma Cell Lines.

We describe the development, optimization, and validation of 384-well growth inhibition assays for six patient-derived melanoma cell lines (PDMCLs), three wild type (WT) for BRAF and three with V600E-BRAF mutations. We conducted a pilot drug combination (DC) high-throughput screening (HTS) of 45 pairwise 4×4 DC matrices prepared from 10 drugs in the PDMCL assays: two B-Raf inhibitors (BRAFi), a MEK inhibitor (MEKi), and a methylation agent approved for melanoma; cytotoxic topoisomerase II and DNA methyltransferase chemotherapies; and drugs targeting the base excision DNA repair enzyme APE1 (apurinic/apyrimidinic endonuclease-1/redox effector factor-1), SRC family tyrosine kinases, the heat shock protein 90 (HSP90) molecular chaperone, and histone deacetylases.Pairwise DCs between dasatinib and three drugs approved for melanoma therapy-dabrafenib, vemurafenib, or trametinib-were flagged as synergistic in PDMCLs. Exposure to fixed DC ratios of the SRC inhibitor dasatinib with the BRAFis or MEKis interacted synergistically to increase PDMCL sensitivity to growth inhibition and enhance cytotoxicity independently of PDMCL BRAF status. These DCs synergistically inhibited the growth of mouse melanoma cell lines that either were dabrafenib-sensitive or had acquired resistance to dabrafenib with cross resistance to vemurafenib, trametinib, and dasatinib. Dasatinib DCs with dabrafenib, vemurafenib, or trametinib activated apoptosis and increased cell death in melanoma cells independently of their BRAF status or their drug resistance phenotypes. These preclinical in vitro studies provide a data-driven rationale for the further investigation of DCs between dasatinib and BRAFis or MEKis as candidates for melanoma combination therapies with the potential to improve outcomes and/or prevent or delay the emergence of disease resistance.

Selecting a minimal set of androgen receptor assays for screening chemicals.

Screening certain environmental chemicals for their ability to interact with endocrine targets, including the androgen receptor (AR), is an important global concern. We previously developed a model using a battery of eleven in vitro AR assays to predict in vivo AR activity. Here we describe a revised mathematical modeling approach that also incorporates data from newly available assays and demonstrate that subsets of assays can provide close to the same level of predictivity. These subset models are evaluated against the full model using 1820 chemicals, as well as in vitro and in vivo reference chemicals from the literature. Agonist batteries of as few as six assays and antagonist batteries of as few as five assays can yield balanced accuracies of 95% or better relative to the full model. Balanced accuracy for predicting reference chemicals is 100%. An approach is outlined for researchers to develop their own subset batteries to accurately detect AR activity using assays that map to the pathway of key molecular and cellular events involved in chemical-mediated AR activation and transcriptional activity. This work indicates in vitro bioactivity and in silico predictions that map to the AR pathway could be used in an integrated approach to testing and assessment for identifying chemicals that interact directly with the mammalian AR.

One-electron oxidation of ds(5'-GGG-3') and ds(5'-G(8OG)G-3') and the nature of hole distribution: a density functional theory (DFT) study.

Of particular interest in radiation-induced charge transfer processes in DNA is the extent of hole localization immediately after ionization and subsequent relaxation. To address this, we considered double stranded oligomers containing guanine (G) and 8-oxoguanine (8OG), i.e., ds(5'-GGG-3') and ds(5'-G8OGG-3') in B-DNA conformation. Using DFT, we calculated a variety of properties, viz., vertical and adiabatic ionization potentials, spin density distributions in oxidized stacks, solvent and solute reorganization energies and one-electron oxidation potential (E0) in the aqueous phase. Calculations for the vertical state of the -GGG- cation radical showed that the spin was found mainly (67%) on the middle G. However, upon relaxation to the adiabatic -GGG- cation radical, the spin localized (96%) on the 5'-G, as observed in experiments. Hole localizations on the middle G and 3'-G were higher in energy by 0.5 kcal mol-1 and 0.4 kcal mol-1, respectively, than that of 5'-G. In the -G8OGG- cation radical, the spin localized only on the 8OG in both vertical and adiabatic states. The calculated vertical ionization potentials of -GGG- and -G8OGG- stacks were found to be lower than that of the vertical ionization potential of a single G in DNA. The calculated E0 values of -GGG- and -G8OGG- stacks are 1.15 and 0.90 V, respectively, which owing to stacking effects are substantially lower than the corresponding experimental E0 values of their monomers (1.49 and 1.18 V, respectively). SOMO to HOMO level switching is observed in these oxidized stacks. Consequently, our calculations predict that local double oxidations in DNA will form triplet diradical states, which are especially significant for high LET radiations.

Pharmacokinetics, pharmacodynamics and safety of the inverse retinoic acid-related orphan receptor γ agonist AZD0284.

AIMS: Retinoic acid-related orphan receptor γ (RORγ), a master regulator of T-helper 17 (Th17) cell function and differentiation, is an attractive target for treatment of Th17-driven diseases. This first-in-human study aimed to investigate the pharmacokinetics, pharmacodynamics, safety and tolerability of the inverse RORγ agonist AZD0284. METHODS: We conducted a phase I, randomized, single-blind, placebo-controlled, two-part, first-in-human study with healthy subjects receiving single (4-238 mg) or multiple (12-100 mg) oral doses of AZD0284 or placebo after overnight fasting. Subjects in the one single dose cohort additionally received a single dose of AZD0284 after a high-calorie meal. AZD0284 plasma concentrations, as well as inhibition of ex vivo-stimulated interleukin (IL)-17A release in whole blood, were frequently measured after both single and multiple dosing. RESULTS: Eighty-three men participated in the study. AZD0284 was absorbed rapidly into plasma after oral dosing and exhibited a terminal half-life of 13-16 hours. Both the area under the concentration-time curve (AUC) and maximum concentration (Cmax ) increased subproportionally with increasing dose (95% confidence intervals of slope parameter were 0.71-0.84 and 0.72-0.88 for AUC and Cmax , respectively). Food intake delayed the absorption of AZD0284 but did not affect the overall exposure or half-life. AZD0284 showed dose-dependent reduction of ex vivo-stimulated IL-17A release after both single and multiple doses. No significant safety concerns were identified in the study. CONCLUSIONS: AZD0284 was well tolerated, rapidly and dose-dependently absorbed, and reduced stimulated IL-17A release after single and multiple dosing. The results of this study support further clinical development of AZD0284.

Maximizing the Value of Cancer Drug Screening in Multicellular Tumor Spheroid Cultures: A Case Study in Five Head and Neck Squamous Cell Carcinoma Cell Lines.

With approval rates <5% and the probability of success in oncology clinical trials of 3.4%, more physiologically relevant in vitro three-dimensional models are being deployed during lead generation to select better drug candidates for solid tumors. Multicellular tumor spheroids (MCTSs) resemble avascular tumor nodules, micrometastases, or the intervascular regions of large solid tumors with respect to morphology, cell-cell and cell-extracellular matrix contacts, and volume growth kinetics. MCTSs develop gradients of nutrient and oxygen concentration resulting in diverse microenvironments with differential proliferation and drug distribution zones. We produced head and neck squamous cell carcinoma (HNSCC) MCTSs in 384-well U-bottom ultra-low-attachment microtiter plates and used metabolic viability and imaging methods to measure morphologies, growth phenotypes and the effects of 19 anticancer drugs. We showed that cell viability measurements underestimated the impact of drug exposure in HNSCC MCTS cultures, but that incorporating morphology and dead-cell staining analyses increased the number of drugs judged to have substantially impacted MCTS cultures. A cumulative multiparameter drug impact score enabled us to stratify MCTS drug responses into high-, intermediate-, and low-impact tiers, and maximized the value of these more physiologically relevant tumor cultures. It is conceivable that the viable cells present in MCTS cultures after drug exposure arise from drug-resistant populations that could represent a source of drug failure and recurrence. Long-term monitoring of treated MCTS cultures could provide a strategy to determine whether these drug-resistant populations represent circumstances where tumor growth is delayed and may ultimately give rise to regrowth.

Discovery of Non-peptide Small Molecule Allosteric Modulators of the Src-family Kinase, Hck.

The eight mammalian Src-family tyrosine kinases are dynamic, multi-domain structures, which adopt distinct "open" and "closed" conformations. In the closed conformation, the regulatory SH3 and SH2 domains pack against the back of the kinase domain, providing allosteric control of kinase activity. Small molecule ligands that engage the regulatory SH3-SH2 region have the potential to modulate Src-family kinase activity for therapeutic advantage. Here we describe an HTS-compatible fluorescence polarization assay to identify small molecules that interact with the unique-SH3-SH2-linker (U32L) region of Hck, a Src-family member expressed exclusively in cells of myeloid lineage. Hck has significant potential as a drug target in acute myeloid leukemia, an aggressive form of cancer with substantial unmet clinical need. The assay combines recombinant Hck U32L protein with a fluorescent probe peptide that binds to the SH3 domain in U32L, resulting in an increased FP signal. Library compounds that interact with the U32L protein and interfere with probe binding reduce the FP signal, scoring as hits. Automated 384-well high-throughput screening of 60,000 compounds yielded Z'-factor coefficients > 0.7 across nearly 200 assay plates, and identified a series of hit compounds with a shared pyrimidine diamine substructure. Surface plasmon resonance assays confirmed direct binding of hit compounds to the Hck U32L target protein as well as near-full-length Hck. Binding was not observed with the individual SH3 and SH2 domains, demonstrating that these compounds recognize a specific three-dimensional conformation of the regulatory regions. This conclusion is supported by computational docking studies, which predict ligand contacts with a pocket formed by the juxtaposition of the SH3 domain, the SH3-SH2 domain connector, and the SH2-kinase linker. Each of the four validated hits stimulated recombinant, near-full-length Hck activity in vitro, providing evidence for allosteric effects on the kinase domain. These results provide a path to discovery and development of chemical scaffolds to target the regulatory regions of Hck and other Src family kinases as a new approach to pharmacological kinase control.

Comprehensive model for X-ray-induced damage in protein crystallography.

Acquisition of X-ray crystallographic data is always accompanied by structural degradation owing to the absorption of energy. The application of high-fluency X-ray sources to large biomolecules has increased the importance of finding ways to curtail the onset of X-ray-induced damage. A significant effort has been under way with the aim of identifying strategies for protecting protein structure. A comprehensive model is presented that has the potential to explain, both qualitatively and quantitatively, the structural changes induced in crystalline protein at ∼100 K. The first step is to consider the qualitative question: what are the radiation-induced intermediates and expected end products? The aim of this paper is to assist in optimizing these strategies through a fundamental understanding of radiation physics and chemistry, with additional insight provided by theoretical calculations performed on the many schemes presented.

Confirmation of Selected Synergistic Cancer Drug Combinations Identified in an HTS Campaign and Exploration of Drug Efflux Transporter Contributions to the Mode of Synergy.

Systematic unbiased high-throughput screening (HTS) of drug combinations (DCs) in well-characterized tumor cell lines is a data-driven strategy to identify novel DCs with potential to be developed into effective therapies. Four DCs from a DC HTS campaign were selected for confirmation; only one appears in clinicaltrials.gov and limited preclinical in vitro data indicates that the drug pairs interact synergistically. Nineteen DC-tumor cell line sets were confirmed to interact synergistically in three pharmacological interaction models. We developed an imaging assay to quantify accumulation of the ABCG2 efflux transporter substrate Hoechst. Gefitinib and raloxifene enhanced Hoechst accumulation in ABCG2 (BCRP)-expressing cells, consistent with inhibition of ABCG2 efflux. Both drugs also inhibit ABCB1 efflux. Mitoxantrone, daunorubicin, and vinorelbine are substrates of one or more of the ABCG2, ABCB1, or ABCC1 efflux transporters expressed to varying extents in the selected cell lines. Interactions between ABC drug efflux transporter inhibitors and substrates may have contributed to the observed synergy; however, other mechanisms may be involved. Novel synergistic DCs identified by HTS were confirmed in vitro, and plausible mechanisms of action studied. Similar approaches may justify the testing of novel HTS-derived DCs in mouse xenograft human cancer models and support the clinical evaluation of effective in vivo DCs in patients.

Implementation of the NCI-60 Human Tumor Cell Line Panel to Screen 2260 Cancer Drug Combinations to Generate >3 Million Data Points Used to Populate a Large Matrix of Anti-Neoplastic Agent Combinations (ALMANAC) Database.

Animal and clinical studies demonstrate that cancer drug combinations (DCs) are more effective than single agents. However, it is difficult to predict which DCs will be more efficacious than individual drugs. Systematic DC high-throughput screening (HTS) of 100 approved drugs in the National Cancer Institute's panel of 60 cancer cell lines (NCI-60) produced data to help select DCs for further consideration. We miniaturized growth inhibition assays into 384-well format, increased the fetal bovine serum amount to 10%, lengthened compound exposure to 72 h, and used a homogeneous detection reagent. We determined the growth inhibition 50% values of individual drugs across 60 cell lines, selected drug concentrations for 4 × 4 DC matrices (DCMs), created DCM master and replica daughter plate sets, implemented the HTS, quality control reviewed the data, and analyzed the results. A total of 2620 DCMs were screened in 60 cancer cell lines to generate 3.04 million data points for the NCI ALMANAC (A Large Matrix of Anti-Neoplastic Agent Combinations) database. We confirmed in vitro a synergistic drug interaction flagged in the DC HTS between the vinca-alkaloid microtubule assembly inhibitor vinorelbine (Navelbine) tartrate and the epidermal growth factor-receptor tyrosine kinase inhibitor gefitinib (Iressa) in the SK-MEL-5 melanoma cell line. Seventy-five percent of the DCs examined in the screen are not currently in the clinical trials database. Selected synergistic drug interactions flagged in the DC HTS described herein were subsequently confirmed by the NCI in vitro, evaluated mechanistically, and were shown to have greater than single-agent efficacy in mouse xenograft human cancer models. Enrollment is open for two clinical trials for DCs that were identified in the DC HTS. The NCI ALMANAC database therefore constitutes a valuable resource for selecting promising DCs for confirmation, mechanistic studies, and clinical translation.

Pharmacodynamic biomarkers and differential effects of TNF- and GM-CSF-targeting biologics in rheumatoid arthritis.

AIM: The aim of our study was to identify pharmacodynamic biomarkers and assess differential effects of tumor necrosis factor (TNF)- and non-TNF-targeting agents on rheumatoid arthritis (RA) patients with an inadequate response to anti-TNF agents (anti-TNF-IR) in comparison with biologic-naïve patients. METHODS: EARTH EXPLORER 2, a phase IIb trial, evaluated golimumab, an anti-TNF antibody, and mavrilimumab, an granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor antibody, in disease-modifying antirheumatic drug (DMARD)-IR and anti-TNF-IR patients. Our current study assessed peripheral protein markers and gene expression levels in association with clinical response post-treatment in two disease strata. RESULTS: Serum proteomics results indicated the existence of specific pharmacodynamic markers for golimumab and mavrilimumab, regardless of prior anti-TNF treatment. In contrast, both antibodies induced early and sustained suppression of RA disease markers, including interleukin (IL)-6, C-reactive protein, IL2RA, and matrix metalloproteinase 1, in DMARD-IR patients. Golimumab-induced early changes rapidly returned toward baseline concentrations in anti-TNF-IR patients, whereas mavrilimumab-induced changes were maintained through to day 169. RNA sequencing demonstrated gene expression changes at day 169 after administration of mavrilimumab but not golimumab in anti-TNF-IR patients. Additionally, receiver operating characteristic curve and regression analysis showed the association of early IL-6 change and subsequent clinical responses to golimumab in anti-TNF-IR patients. CONCLUSION: Our results revealed golimumab- and mavrilimumab-specific pharmacodynamic biomarkers, and demonstrated differential biomarker-treatment relationships in anti-TNF-IR and DMARD-IR patients, respectively. Early IL-6 change after anti-TNF antibody treatment may be a potential predictive biomarker for selection of different treatment regimens in anti-TNF-IR patients.

High Content Screening Characterization of Head and Neck Squamous Cell Carcinoma Multicellular Tumor Spheroid Cultures Generated in 384-Well Ultra-Low Attachment Plates to Screen for Better Cancer Drug Leads.

Multicellular tumor spheroid (MCTS) cultures represent more physiologically relevant in vitro cell tumor models that recapitulate the microenvironments and cell-cell or cell-extracellular matrix interactions which occur in solid tumors. We characterized the morphologies, viability, and growth behaviors of MCTSs produced by 11 different head and neck squamous cell carcinoma (HNSCC) cell lines seeded into and cultured in ultra-low attachment microtiter plates (ULA-plates) over extended periods of time. HNSCC MCTS cultures developed microenvironments, which resulted in differences in proliferation rates, metabolic activity, and mitochondrial functional activity between cells located in the outer layers of the MCTS and cells in the interior. HNSCC MCTS cultures exhibited drug penetration and distribution gradients and some developed necrotic cores. Perhaps the most profound effect of culturing HNSCC cell lines in MCTS cultures was their dramatically altered and varied growth phenotypes. Instead of the exponential growth that are characteristic of two-dimensional HNSCC growth inhibition assays, some MCTS cultures displayed linear growth rates, categorized as rapid, moderate, or slow, dormant MCTSs remained viable but did not grow, and some MCTSs exhibited death phenotypes that were either progressive and slow or rapid. The ability of MCTS cultures to develop microenvironments and to display a variety of different growth phenotypes provides in vitro models that are more closely aligned with solid tumors in vivo. We anticipate that the implementation MCTS models to screen for new cancer drugs for solid tumors like HNSCC will produce leads that will translate better in in vivo animal models and patients.

High-Content Screening Campaign to Identify Compounds That Inhibit or Disrupt Androgen Receptor-Transcriptional Intermediary Factor 2 Protein-Protein Interactions for the Treatment of Prostate Cancer.

Twenty percent of prostate cancer (PCa) patients develop a noncurable drug-resistant form of the disease termed castration-resistant prostate cancer (CRPC). Overexpression of Androgen Receptor (AR) coactivators such as transcriptional intermediary factor 2 (TIF2) is associated with poor CRPC patient outcomes. We describe the implementation of the AR-TIF2 protein-protein interaction biosensor (PPIB) assay in a high-content screening (HCS) campaign of 143,535 compounds. The assay performed robustly and reproducibly and enabled us to identify compounds that inhibited dihydrotestosterone (DHT)-induced AR-TIF2 protein-protein interaction (PPI) formation or disrupted preexisting AR-TIF2 PPIs. We used multiparameter HCS data z-scores to identify and deprioritize cytotoxic or autofluorescent outliers and confirmed the resulting qualified actives in triplicate. None of the confirmed AR-TIF2 PPIB inhibitors/disruptors exhibited activity in a p53-hDM2 PPIB counter screen, indicating that they were unlikely to be either nonselective PPI inhibitors or to interfere with the biosensor assay format. However, eight confirmed AR-TIF2 PPIB actives also inhibited the glucocorticoid receptor (GR) nuclear translocation counter screen by >50%. These compounds were deprioritized because they either lacked AR specificity/selectivity, or they inhibited a shared component of the AR and GR signaling pathways. Twenty-nine confirmed AR-TIF2 PPIB actives also inhibited the AR nuclear localization counter screen, suggesting that they might indirectly inhibit the AR-TIF2 PPIB assay rather than directly blocking/disrupting PPIs. A total of 62.2% of the confirmed actives inhibited the DHT-induced AR-TIF2 PPI formation in a concentration-dependent manner with IC50s < 40 µM, and 59.4% also disrupted preexisting AR-TIF2 PPI complexes. Overall, the hit rate for the AR-TIF2 PPIB HCS campaign was 0.12%, and most hits inhibited AR-TIF2 PPI formation and disrupted preexisting AR-TIF2 complexes with similar AR-red fluorescent protein distribution phenotypes. Further secondary and tertiary hit characterization assays are underway to select AR-TIF2 PPI inhibitor/disruptor hits suitable for medicinal chemistry lead optimization and development into novel PCa/CRPC therapeutics.

Mavrilimumab, a Fully Human Granulocyte-Macrophage Colony-Stimulating Factor Receptor α Monoclonal Antibody: Long-Term Safety and Efficacy in Patients With Rheumatoid Arthritis.

OBJECTIVE: Mavrilimumab, a human monoclonal antibody, targets granulocyte-macrophage colony-stimulating factor receptor α. We undertook to determine the long-term safety and efficacy of mavrilimumab in rheumatoid arthritis patients in 2 phase IIb studies (1071 and 1107) and in 1 open-label extension study (ClinicalTrials.gov identifier: NCT01712399). METHODS: In study 1071, patients with an inadequate response to disease-modifying antirheumatic drugs (DMARDs) received mavrilimumab (30, 100, or 150 mg) or placebo every other week plus methotrexate. In study 1107, patients with an inadequate response to anti-tumor necrosis factor agents and/or DMARDs received 100 mg mavrilimumab every other week or 50 mg golimumab every 4 weeks plus methotrexate. Patients entering the open-label extension study received 100 mg mavrilimumab every other week plus methotrexate. Long-term safety and efficacy of mavrilimumab were assessed. RESULTS: A total of 442 patients received mavrilimumab (14 of 245 patients from study 1071, 9 of 70 patients from study 1107, and 52 of 397 patients from the open-label extension study discontinued mavrilimumab treatment throughout the studies). The cumulative safety exposure was 899 patient-years; the median duration of mavrilimumab treatment was 2.5 years (range 0.1-3.3 years). The most common treatment-emergent adverse events (AEs) were nasopharyngitis (n = 69; 7.68 per 100 patient-years) and bronchitis (n = 51; 5.68 per 100 patient-years). At weeks 74 and 104, 3.5% and 6.2% of patients, respectively, demonstrated reduction in forced expiratory volume in 1 second, while 2.9% and 3.4% of patients, respectively, demonstrated reduction in forced vital capacity (>20% reduction from baseline to <80% predicted). Most pulmonary changes were transient and only infrequently associated with AEs. Mavrilimumab at 100 mg every other week demonstrated sustained efficacy; at week 122, 65.0% of patients achieved a Disease Activity Score in 28 joints using the C-reactive protein level (DAS28-CRP) of <3.2, and 40.6% of patients achieved a DAS28-CRP of <2.6. CONCLUSION: Long-term treatment with mavrilimumab maintained response and was well-tolerated with no increased incidence of treatment-emergent AEs. Safety data were comparable with those from both phase IIb qualifying studies.

A Randomized Phase IIb Study of Mavrilimumab and Golimumab in Rheumatoid Arthritis.

OBJECTIVE: This 24-week, phase IIb, double-blind study was undertaken to evaluate the efficacy and safety of mavrilimumab (a monoclonal antibody to granulocyte-macrophage colony-stimulating factor receptor α) and golimumab (a monoclonal antibody to tumor necrosis factor [anti-TNF]) in patients with rheumatoid arthritis (RA) who have had an inadequate response to disease-modifying antirheumatic drugs (DMARDs) (referred to as DMARD-IR) and/or inadequate response to other anti-TNF agents (referred to as anti-TNF-IR). METHODS: Patients with active RA and a history of DMARD-IR (≥1 failed regimen) or DMARD-IR (≥1 failed regimen) and anti-TNF-IR (1-2 failed regimens) were randomized 1:1 to receive either mavrilimumab 100 mg subcutaneously every other week or golimumab 50 mg subcutaneously every 4 weeks alternating with placebo every 4 weeks, administered concomitantly with methotrexate. The primary end points were the American College of Rheumatology 20% improvement (ACR20), 50% improvement, and 70% improvement response rates at week 24, percentage of patients achieving a Disease Activity Score in 28 joints using C-reactive protein level (DAS28-CRP) of <2.6 at week 24, percentage of patients with a score improvement of >0.22 on the Health Assessment Questionnaire (HAQ) disability index (DI) at week 24, and safety/tolerability measures. This study was not powered to formally compare the 2 treatments. RESULTS: At week 24, differences in the ACR20, ACR50, and ACR70 response rates between the mavrilimumab treatment group (n = 70) and golimumab treatment group (n = 68) were as follows: in all patients, -3.5% (90% confidence interval [90% CI] -16.8, 9.8), -8.6% (90% CI -22.0, 4.8), and -9.8% (90% CI -21.1, 1.4), respectively; in the anti-TNF-IR group, 11.1% (90% CI -7.8, 29.9), -8.7% (90% CI -28.1, 10.7), and -0.7% (90% CI -18.0, 16.7), respectively. Differences in the percentage of patients achieving a DAS28-CRP of <2.6 at week 24 between the mavrilimumab and golimumab groups were -11.6% (90% CI -23.2, 0.0) in all patients, and -4.0% (90% CI -20.9, 12.9) in the anti-TNF-IR group. The percentage of patients achieving a >0.22 improvement in the HAQ DI score at week 24 was similar between the treatment groups. Treatment-emergent adverse events were reported in 51.4% of mavrilimumab-treated patients and 42.6% of golimumab-treated patients. No deaths were reported, and no specific safety signals were identified. CONCLUSION: The findings of this study demonstrate the clinical efficacy of both treatments, mavrilimumab at a dosage of 100 mg every other week and golimumab at a dosage of 50 mg every 4 weeks, in patients with RA. Both regimens were well-tolerated in patients who had shown an inadequate response to DMARDs and/or other anti-TNF agents.

High-Content Screening Comparison of Cancer Drug Accumulation and Distribution in Two-Dimensional and Three-Dimensional Culture Models of Head and Neck Cancer.

High cancer drug development attrition rates have provoked considerable debate about whether the two-dimensional tumor growth inhibition high-throughput screening assays used in pre-clinical lead discovery adequately reflect solid tumor complexity. We used automated high-content screening image acquisition and analysis methods to compare fluorescent drug uptake, accumulation, and distribution in Cal33 and FaDu head and neck cancer (HNC) monolayer and multicellular tumor spheroid (MCTS) models. Ellipticine, idarubicin, daunorubicin, and doxorubicin were studied because of their fluorescent properties and broad anti-tumor activities. HNC MCTSs were generated in 384-well ultra-low attachment plates where compound exposure, image acquisition, and analysis could be performed in situ. Fluorescent drug accumulation in Cal33 monolayer and MCTS cultures was linear with respect to concentration, and appeared to achieve steady-state levels within 10-15 min of drug exposure, which were maintained through 30-45 min. Drug accumulation in monolayers was independent of cell number and/or density, and every cell achieved uniform drug concentrations. In MCTSs, however, drug accumulation increased as the number of cells and sizes of the MCTSs became bigger. Drugs exhibited restricted penetration and distribution gradients, accumulating preferentially in cells in the outer layers of MCTSs relative to those in the inner cores. Cal33 monolayers were 6-, 20-, 10-, and 16-fold more sensitive than MCTSs to growth inhibition by ellipticine, idarubicin, daunorubicin, and doxorubicin, respectively. In Cal33 MCTSs exposed to ellipticine or doxorubicin for 24 h, MCTSs were smaller and although they still exhibited drug penetration and distribution gradients, the fluorescent intensity difference between outer and inner cells was reduced. After a 24 h exposure, both drugs had penetrated throughout FaDu MCTSs, consistent with drug-induced death of peripheral cell layers enhancing drug penetration. The increased resistance of MCTS cultures and their ability to recapitulate drug penetration and distribution gradients argues strongly for the deployment of these more physiological models in cancer lead discovery. MCTSs have the potential to enhance the correlation between in vitro potencies and in vivo efficacy, and ultimately may lead to improved cancer drug approval rates.