Discovery of Monovalent Direct Degraders of BRD4 that Act via the Recruitment of DCAF11.

Targeted protein degradation (TPD) using the ubiquitin proteasome system (UPS) is a rapidly growing drug discovery modality to eliminate pathogenic proteins. Strategies for TPD have focused on heterobifunctional degraders that often suffer from poor drug-like properties, and molecular glues that rely on serendipitous discovery. Monovalent "direct" degraders represent an alternative approach, in which small molecules bind to a target protein and induce degradation of that protein through the recruitment of an E3 ligase complex. Using an ultra-high throughput cell-based screening platform, degraders of the bromodomain extraterminal protein BRD4 were identified and optimized to yield a lead compound, PLX-3618. In this paper, we demonstrate that PLX-3618 elicited UPS-mediated selective degradation of BRD4, resulting in potent antitumor activity in vitro and in vivo. Characterization of the degradation mechanism identified DCAF11 as the E3 ligase required for PLX-3618-mediated degradation of BRD4. Protein-protein interaction studies verified a BRD4:PLX-3618:DCAF11 ternary complex, and mutational studies provided further insights into the DCAF11-mediated degradation mechanism. Collectively, these results demonstrate the discovery and characterization of a novel small molecule that selectively degrades BRD4 through the recruitment of the E3 substrate receptor, DCAF11, and promotes potent antitumor activity in vivo.

Discovery of Monovalent Direct Degraders of BRD4 That Act Via the Recruitment of DCAF11.

Targeted protein degradation (TPD) using the ubiquitin proteasome system (UPS) is a rapidly growing drug discovery modality to eliminate pathogenic proteins. Strategies for TPD have focused on heterobifunctional degraders that often suffer from poor drug-like properties, and molecular glues that rely on serendipitous discovery. Monovalent "direct" degraders represent an alternative approach, in which small molecules bind to a target protein and induce degradation of that protein through the recruitment of an E3 ligase complex. Using an ultra-high throughput cell-based screening platform, degraders of the bromodomain extra-terminal (BET) protein BRD4 were identified and optimized to yield a lead compound, PLX-3618. In this paper, we demonstrate that PLX-3618 elicited UPS-mediated selective degradation of BRD4, resulting in potent anti-tumor activity in vitro and in vivo. Characterization of the degradation mechanism identified DCAF11 as the E3 ligase required for PLX-3618-mediated degradation of BRD4. Protein-protein interaction studies verified a BRD4:PLX-3618:DCAF11 ternary complex, and mutational studies provided further insights into the DCAF11-mediated degradation mechanism. Collectively, these results demonstrate the discovery and characterization of a novel small molecule that selectively degrades BRD4 through the recruitment of the E3 substrate receptor, DCAF11, and promotes potent anti-tumor activity in vivo.

eIF4A controls translation of estrogen receptor alpha and is a therapeutic target in advanced breast cancer.

The majority of human breast cancers are dependent on hormone-stimulated estrogen receptor alpha (ER) and are sensitive to its inhibition. Treatment resistance arises in most advanced cancers due to genetic alterations that promote ligand independent activation of ER itself or ER target genes. Whereas re-targeting of the ER ligand binding domain (LBD) with newer ER antagonists can work in some cases, these drugs are largely ineffective in many genetic backgrounds including ER fusions that lose the LBD or in cancers that hyperactivate ER targets. By identifying the mechanism of ER translation, we herein present an alternative strategy to target ER and difficult to treat ER variants. We find that ER translation is cap-independent and mTOR inhibitor insensitive, but dependent on 5' UTR elements and sensitive to pharmacologic inhibition of the translation initiation factor eIF4A, an mRNA helicase. EIF4A inhibition rapidly reduces expression of ER and short-lived targets of ER such as cyclin D1 and other components of the cyclin D-CDK complex in breast cancer cells. These effects translate into suppression of growth of a variety of ligand-independent breast cancer models including those driven by ER fusion proteins that lack the ligand binding site. The efficacy of eIF4A inhibition is enhanced when it is combined with fulvestrant-an ER degrader. Concomitant inhibition of ER synthesis and induction of its degradation causes synergistic and durable inhibition of ER expression and tumor growth. The clinical importance of these findings is confirmed by results of an early clinical trial (NCT04092673) of the selective eIF4A inhibitor zotatifin in patients with estrogen receptor positive metastatic breast cancer. Multiple clinical responses have been observed on combination therapy including durable regressions. These data suggest that eIF4A inhibition could be a useful new strategy for treating advanced ER+ breast cancer.

Zotatifin, an eIF4A-Selective Inhibitor, Blocks Tumor Growth in Receptor Tyrosine Kinase Driven Tumors.

Oncoprotein expression is controlled at the level of mRNA translation and is regulated by the eukaryotic translation initiation factor 4F (eIF4F) complex. eIF4A, a component of eIF4F, catalyzes the unwinding of secondary structure in the 5'-untranslated region (5'-UTR) of mRNA to facilitate ribosome scanning and translation initiation. Zotatifin (eFT226) is a selective eIF4A inhibitor that increases the affinity between eIF4A and specific polypurine sequence motifs and has been reported to inhibit translation of driver oncogenes in models of lymphoma. Here we report the identification of zotatifin binding motifs in the 5'-UTRs of HER2 and FGFR1/2 Receptor Tyrosine Kinases (RTKs). Dysregulation of HER2 or FGFR1/2 in human cancers leads to activation of the PI3K/AKT and RAS/ERK signaling pathways, thus enhancing eIF4A activity and promoting the translation of select oncogenes that are required for tumor cell growth and survival. In solid tumor models driven by alterations in HER2 or FGFR1/2, downregulation of oncoprotein expression by zotatifin induces sustained pathway-dependent anti-tumor activity resulting in potent inhibition of cell proliferation, induction of apoptosis, and significant in vivo tumor growth inhibition or regression. Sensitivity of RTK-driven tumor models to zotatifin correlated with high basal levels of mTOR activity and elevated translational capacity highlighting the unique circuitry generated by the RTK-driven signaling pathway. This dependency identifies the potential for rational combination strategies aimed at vertical inhibition of the PI3K/AKT/eIF4F pathway. Combination of zotatifin with PI3K or AKT inhibitors was beneficial across RTK-driven cancer models by blocking RTK-driven resistance mechanisms demonstrating the clinical potential of these combination strategies.

1-Aminomethyl SAR in a novel series of flavagline-inspired eIF4A inhibitors: Effects of amine substitution on cell potency and in vitro PK properties.

Flavaglines such as silvestrol (1) and rocaglamide (2) constitute an interesting class of natural products with promising anticancer activities. Their mode of action is based on inhibition of eukaryotic initiation factor 4A (eIF4A) dependent translation through formation of a stable ternary complex with eIF4A and mRNA, thus blocking ribosome scanning. Herein we describe initial SAR studies in a novel series of 1-aminomethyl substituted flavagline-inspired eIF4A inhibitors. We discovered that a variety of N-substitutions at the 1-aminomethyl group are tolerated, making this position pertinent for property and ADME profile tuning. The findings presented herein are relevant to future drug design efforts towards novel eIF4A inhibitors with drug-like properties.

Targeting Oncogene mRNA Translation in B-Cell Malignancies with eFT226, a Potent and Selective Inhibitor of eIF4A.

The PI3K/AKT/mTOR pathway is often activated in lymphoma through alterations in PI3K, PTEN, and B-cell receptor signaling, leading to dysregulation of eIF4A (through its regulators, eIF4B, eIF4G, and PDCD4) and the eIF4F complex. Activation of eIF4F has a direct role in tumorigenesis due to increased synthesis of oncogenes that are dependent on enhanced eIF4A RNA helicase activity for translation. eFT226, which inhibits translation of specific mRNAs by promoting eIF4A1 binding to 5'-untranslated regions (UTR) containing polypurine and/or G-quadruplex recognition motifs, shows potent antiproliferative activity and significant in vivo efficacy against a panel of diffuse large B-cell lymphoma (DLBCL), and Burkitt lymphoma models with ≤1 mg/kg/week intravenous administration. Evaluation of predictive markers of sensitivity or resistance has shown that activation of eIF4A, mediated by mTOR signaling, correlated with eFT226 sensitivity in in vivo xenograft models. Mutation of PTEN is associated with reduced apoptosis in vitro and diminished efficacy in vivo in response to eFT226. In models evaluated with PTEN loss, AKT was stimulated without a corresponding increase in mTOR activation. AKT activation leads to the degradation of PDCD4, which can alter eIF4F complex formation. The association of eFT226 activity with PTEN/PI3K/mTOR pathway regulation of mRNA translation provides a means to identify patient subsets during clinical development.

Strategic Diastereoselective C1 Functionalization in the Aza-Rocaglamide Scaffold toward Natural Product-Inspired eIF4A Inhibitors.

Rocaglates, rocaglamides, and related flavagline natural products exert their remarkable anticancer activity through inhibition of eukaryotic initiation factor 4A (eIF4A) but generally display suboptimal drug-like properties. In our efforts to identify potent drug-like eIF4A inhibitors, we developed synthetic strategies for diastereoselectively functionalizing the C1 position of aza-rocaglamide scaffolds (cf. 14 and 18), which proceed via retention or inversion of configuration at C1 depending on the C2 substituent (cf. 15 and 21) and ultimately enabled the discovery of novel and potent eIF4A inhibitors such as 25.

Design of Development Candidate eFT226, a First in Class Inhibitor of Eukaryotic Initiation Factor 4A RNA Helicase.

Dysregulation of protein translation is a key driver for the pathogenesis of many cancers. Eukaryotic initiation factor 4A (eIF4A), an ATP-dependent DEAD-box RNA helicase, is a critical component of the eIF4F complex, which regulates cap-dependent protein synthesis. The flavagline class of natural products (i.e., rocaglamide A) has been shown to inhibit protein synthesis by stabilizing a translation-incompetent complex for select messenger RNAs (mRNAs) with eIF4A. Despite showing promising anticancer phenotypes, the development of flavagline derivatives as therapeutic agents has been hampered because of poor drug-like properties as well as synthetic complexity. A focused effort was undertaken utilizing a ligand-based design strategy to identify a chemotype with optimized physicochemical properties. Also, detailed mechanistic studies were undertaken to further elucidate mRNA sequence selectivity, key regulated target genes, and the associated antitumor phenotype. This work led to the design of eFT226 (Zotatifin), a compound with excellent physicochemical properties and significant antitumor activity that supports clinical development.

Incidence of health care-associated extended-spectrum ß-lactamase-positive patients before and after discontinuation of contact precautions.

BACKGROUND: Isolation of patients with multidrug-resistant organisms has been recommended in several guidelines. Recent evidence has suggested potential negative effects of isolation on patient well-being and facility throughput. Published literature shows a difference in transmission risk of extended-spectrum ß-lactamase (ESBL)-producing organisms, suggesting that contact precautions may not be necessary for all ESBL-positive organisms. METHODS: Incidence rates of health care-associated ESBL organisms were measured before and after eliminating the use of contact precautions for patients with only ESBL-positive organisms. The National Healthcare Safety Network surveillance methodology was used to measure incidence. Surgical site infections and carbapenem-resistant Enterobacteriaceae were excluded from the surveillance incidence. RESULTS: The incidence of health care-associated ESBL infections from January 2014 through November 2015 was 3.71 per 10,000 patient days. The incidence from December 2015 through August 2017 was 3.00 per 10,000 patient days. This rate change was statistically significant (P = .022) CONCLUSIONS: This study found that discontinuing the use of contact precautions for patients colonized or infected with ESBL-positive organisms did not lead to an increased rate of health care-associated ESBL-positive infections or colonization.

Reduction in Central Line-Associated Bloodstream Infections Correlated With the Introduction of a Novel Silver-Plated Dressing for Central Venous Catheters and Maintained for 6 Years.

OBJECTIVE: To assess a novel silver-plated dressing (SD) for central venous catheters in comparison to chlorhexidine gluconate-impregnated sponge (CHGIS) dressings in preventing central line-associated bloodstream infections (CLABSIs) in adult intensive care unit (ICU) patients. DESIGN: Retrospective cohort study. SETTING: Tampa General Hospital, an academic medical tertiary care center. PATIENTS: All adult ICU patients of an academic medical tertiary care center from January 2009 to December 2010. MEASUREMENTS AND MAIN RESULTS: A total of 3189 patient records were studied from 7 different ICUs during the 2-year period. Patients received either CHGIS dressings (January 2009-December 2009) or SDs (January 2010-December 2010). Primary outcomes measured were CLABSI rates per 1000 catheter days and ICU length of stay. There were 30 696 catheter days with CHGIS dressings and 31 319 catheter days with SDs. There was a statistically significant decrease in the rate of CLABSI per 1000 catheter days in the SD group, from 2.38 to 1.28 ( P = .001), with an absolute risk reduction of 1.1. There was a significantly lower incidence in the rate of CLABSI per 1000 catheter days in the SD group (incidence rate ratio [IRR] = 0.54, 95% confidence interval [CI]: 0.36-0.80). The relative risk of CLABSI in the SD group was 0.502 (95% CI: 0.340-0.730; P < .001). If SDs are used on all catheters, the decreased rate of CLABSIs observed would calculate to a cost savings of US$4070 to US$39 600 per 1000 catheter days. After successful implementation of the SD, we observed significant reductions in CLABSI rates and a sustained reduction in the subsequent 6 years. CONCLUSION: Use of SDs is associated with a significant decrease in CLABSI rates in adult ICU patients compared to CHGIS dressings, with an estimated cost savings of US$4070 to US$39 600 per 1000 catheter days.

Structure-based Design of Pyridone-Aminal eFT508 Targeting Dysregulated Translation by Selective Mitogen-activated Protein Kinase Interacting Kinases 1 and 2 (MNK1/2) Inhibition.

Dysregulated translation of mRNA plays a major role in tumorigenesis. Mitogen-activated protein kinase interacting kinases (MNK)1/2 are key regulators of mRNA translation integrating signals from oncogenic and immune signaling pathways through phosphorylation of eIF4E and other mRNA binding proteins. Modulation of these key effector proteins regulates mRNA, which controls tumor/stromal cell signaling. Compound 23 (eFT508), an exquisitely selective, potent dual MNK1/2 inhibitor, was designed to assess the potential for control of oncogene signaling at the level of mRNA translation. The crystal structure-guided design leverages stereoelectronic interactions unique to MNK culminating in a novel pyridone-aminal structure described for the first time in the kinase literature. Compound 23 has potent in vivo antitumor activity in models of diffuse large cell B-cell lymphoma and solid tumors, suggesting that controlling dysregulated translation has real therapeutic potential. Compound 23 is currently being evaluated in Phase 2 clinical trials in solid tumors and lymphoma. Compound 23 is the first highly selective dual MNK inhibitor targeting dysregulated translation being assessed clinically.

Use of a Parabolic Microphone to Detect Hidden Subjects in Search and Rescue.

INTRODUCTION: This study compares a parabolic microphone to unaided hearing in detecting and comprehending hidden callers at ranges of 322 to 2510 m. METHODS: Eight subjects were placed 322 to 2510 m away from a central listening point. The subjects were concealed, and their calling volume was calibrated. In random order, subjects were asked to call the name of a state for 5 minutes. Listeners with parabolic microphones and others with unaided hearing recorded the direction of the call (detection) and name of the state (comprehension). RESULTS: The parabolic microphone was superior to unaided hearing in both detecting subjects and comprehending their calls, with an effect size (Cohen's d) of 1.58 for detection and 1.55 for comprehension. For each of the 8 hidden subjects, there were 24 detection attempts with the parabolic microphone and 54 to 60 attempts by unaided listeners. At the longer distances (1529-2510 m), the parabolic microphone was better at detecting callers (83% vs 51%; P<0.00001 by χ2) and comprehension (57% vs 12%; P<0.00001). At the shorter distances (322-1190 m), the parabolic microphone offered advantages in detection (100% vs 83%; P=0.000023) and comprehension (86% vs 51%; P<0.00001), although not as pronounced as at the longer distances. CONCLUSIONS: Use of a 66-cm (26-inch) parabolic microphone significantly improved detection and comprehension of hidden calling subjects at distances between 322 and 2510 m when compared with unaided hearing. This study supports the use of a parabolic microphone in search and rescue to locate responsive subjects in favorable weather and terrain.

Decreasing methicillin-resistant Staphylococcus aureus surgical site infections with chlorhexidine and mupirocin.

BACKGROUND: Surgical site infections (SSIs) associated with methicillin-resistant Staphylococcus aureus (MRSA) are a major complication of surgery. This study is part of a large infection control quality improvement effort to eliminate MRSA SSIs. METHODS: This is an Institutional Review Board-approved, case-control study examining MRSA SSI rates before and after implementation of a facility-wide MRSA SSI prevention protocol in 2007. The protocol involved a 5-day course of intranasal mupirocin and nonrinse 2% chlorhexidine gluconate cloths. RESULTS: In 2006, a total of 39 MRSA SSIs occurred after 9,976 procedures in patients who underwent orthopedic, vascular, cardiac, or neurosurgical procedures (rate = 0.39 per 100 procedures). The highest incidence occurred after cardiac surgery (rate = 1.24/100 procedures), and the lowest occurred after orthopedic surgery (rate = 0.28/100 procedures). In 2007, the MRSA SSI rate decreased to 20 of 9,818 procedures (rate = 0.20/100 procedures) in the 4 categories (P = .016; odds ratio, 1.92). In 2008, the MRSA SSI rate dropped again to 13 of 10,068 procedures (rate = 0.13/100 procedures) in the 4 categories (P = .0003; odds ratio, 3.04). CONCLUSION: A 5-day course of intranasal mupirocin and nonrinse 2% chlorhexidine gluconate cloths may be beneficial in preventing MRSA SSIs in the non-general surgery population.

Early identification and control of carbapenemase-producing Klebsiella pneumoniae, originating from contaminated endoscopic equipment.

Klebsiella producing carbapenemase is an emerging pathogen. We report transmission of this organism by contaminated endoscopic instruments. Quick identification of source, staff education, contact precautions, and emphasis on hand and environmental hygiene led to case control and prevention of outbreak.

Discovery of tricyclic 5,6-dihydro-1H-pyridin-2-ones as novel, potent, and orally bioavailable inhibitors of HCV NS5B polymerase.

A novel series of non-nucleoside small molecules containing a tricyclic dihydropyridinone structural motif was identified as potent HCV NS5B polymerase inhibitors. Driven by structure-based design and building on our previous efforts in related series of molecules, we undertook extensive SAR studies, in which we identified a number of metabolically stable and very potent compounds in genotype 1a and 1b replicon assays. This work culminated in the discovery of several inhibitors, which combined potent in vitro antiviral activity against both 1a and 1b genotypes, metabolic stability, good oral bioavailability, and high C(12) (PO)/EC(50) ratios.

SCAN--a high-throughput assay for detecting small molecule binding to RNA targets.

A novel optical-based high-throughput screening technology has been developed for increasing the rate of discovering chemical leads against RNA targets. SCAN ( Screen for Compounds with Affinity for Nucleic Acids) is an affinity-based assay that identifies small molecules that bind and recognize structured RNA elements. This technology provides the opportunity to conduct high-throughput screening of a new class of targets-RNA. SCAN offers many attractive features including a simple homogeneous format, low screening costs, and the ability to use common laboratory equipment. A SCAN assay was developed for the HCV IRES Loop IIId RNA domain. A high-throughput screen of our entire compound library resulted in the identification of small molecule ligands that bind to Loop IIId. The Z' values were greater than 0.8, showing this to be a robust high-throughput screening assay. A correlation between SCAN EC50 and KD values is reported suggesting the ability to use the assay for compound optimization.

Structure of hepatitis C virus IRES subdomain IIa.

The hepatitis C (HCV) internal ribosome entry site (IRES) element plays a central role in cap-independent translation of the viral genomic RNA. The unique conformation of IRES domain II is critical for 80S ribosomal assembly and initiation of viral translation. Here, the crystal structure of subdomain IIa of the HCV IRES has been determined at 2.3 A resolution, revealing the positions of divalent metal ions and complex inter-strand interactions that stabilize the L-shaped conformation of the RNA. The presence of divalent metal ions was necessary for crystal formation. Magnesium ions occupy specific sites that appear to be critical for the formation of the folded conformation. Subdomain IIa also was crystallized in the presence of strontium, which improved the diffraction quality of the crystals and the ability to identify interactions of the RNA with metal ions and tightly bound water molecules. The hinge region and noncanonical G-U base-pair motifs are stabilized by divalent metal ions and provide unique structural features that are potential interaction sites for small-molecule ligands. The information obtained from the crystal structure provides a basis for structure-guided design of HCV translation inhibitors targeting disruption of ribosomal assembly.

ATLAS--a high-throughput affinity-based screening technology for soluble proteins: technology application using p38 MAP kinase.

Abstract: A general affinity-based screening assay for discovery of lead compounds binding to potential protein drug targets that is based upon protein thermal unfolding and aggregation is described. ATLAS (Any Target Ligand Affinity Screen) (Anadys Pharmaceuticals, Inc., San Diego, CA) is a simple, homogeneous, and high-throughput affinity-based screening technology that can identify compounds that bind and protect the target protein from thermal unfolding, denaturation, and subsequent aggregation. ATLAS detection of thermally unfolded and aggregated hexahistidine [(His)6]-tagged proteins uses time-resolved fluorescence resonance energy transfer between two anti-(His)6 antibodies, labeled with either a donor or acceptor fluorophore, that are simultaneously bound to the aggregated protein. The ATLAS assay is simple to perform and easily automated for screening large compound libraries. The technology is applicable to lead discovery for soluble proteins of known and unknown functions, and particularly for proteins that are difficult to assay functionally. The ATLAS technology has been evaluated using p38 mitogen-activated protein (MAP) kinase as the target protein. Known inhibitors of p38 MAP kinase were examined by ATLAS and a functional assay; the results showed good correlation between the two methods.

Identification of ligand binding by protein stabilization: comparison of ATLAS with biophysical and enzymatic methods.

ATLAS (Any Target Ligand Affinity Screen) (Anadys Pharmaceuticals, Inc., San Diego, CA) is a homogeneous, affinity-based high-throughput screening technology based on protein thermal denaturation and the ability of ligands to bind and stabilize the target protein from unfolding. To further understand the assay sensitivity for the identification of ligands that bind to soluble protein targets, firefly luciferase was chosen to characterize the technology. Luciferase is a multidomain protein with a complex unfolding pathway. Binding of ATP results in a stabilizing conformational rearrangement of the domains. Using luciferase to characterize the ATLAS technology allowed us to evaluate the generality of the screening method for the identification of ligand binding to any target. Luciferase inhibitors identified from functional screens were used to assess the capability of ATLAS to rank order inhibitors. Comparison of the ATLAS 50% effective concentration with other biophysical and biochemical methods offered insight into optimizing ATLAS assay conditions to maximize sensitivity to compound binding and protein stabilization. The results show the importance of characterizing the thermal unfolding and aggregation behavior of the protein to allow the ATLAS screen to be optimally designed.