In breast cancer, estrogen receptor alpha (ERα) positive cancer accounts for approximately 74% of all diagnoses, and in these settings, it is a primary driver of cell proliferation. Treatment of ERα ...positive breast cancer has long relied on endocrine therapies such as selective estrogen receptor modulators, aromatase inhibitors, and selective estrogen receptor degraders (SERDs). The steroid-based anti-estrogen fulvestrant (5), the only approved SERD, is effective in patients who have not previously been treated with endocrine therapy as well as in patients who have progressed after receiving other endocrine therapies. Its efficacy, however, may be limited due to its poor physicochemical properties. We describe the design and synthesis of a series of potent benzothiophene-containing compounds that exhibit oral bioavailability and preclinical activity as SERDs. This article culminates in the identification of LSZ102 (10), a compound in clinical development for the treatment of ERα positive breast cancer.
The respiratory syncytial virus (RSV) L protein is a viral RNA-dependent RNA polymerase that contains multiple enzyme activities required for RSV replication. The RSV L inhibitors described in ...literature are limited by their cytotoxicity or the lack of RSV B subtype coverage. Here, we characterize a new RSV L inhibitor with strong antiviral activity against both RSV A and B subtypes and no detectable cytotoxicity. This compound, AZ-27, was equally active against RSV live viruses and subgenomic replicons and demonstrated advantages over other classes of RSV inhibitors in time-of-addition and cell line dependency studies. Resistance studies identified a dominant mutation in the putative capping enzyme domain of L protein, which conferred strong resistance to the AZ-27 series but not other classes of RSV inhibitors, supporting RSV L protein as the direct target for AZ-27. This novel and broad-spectrum RSV L polymerase inhibitor may pave the way toward an efficacious RSV therapeutic and provide a new tool for interrogation of the L protein function.
Targeting viral polymerases has been a proven and attractive strategy for antiviral drug discovery. Herein we describe our effort in improving the antiviral activity and physical properties of a ...series of benzothienoazepine compounds as respiratory syncytial virus (RSV) RNA polymerase inhibitors. The antiviral activity and spectrum of this class was significantly improved by exploring the amino substitution of the pyridine ring, resulting in the discovery of the most potent RSV A polymerase inhibitors reported to date.
Respiratory syncytial virus (RSV) infects 99% of children by age 2 years and is a leading cause of serious lower respiratory tract infection (LRTI) and infant hospitalization in the United Kingdom. ...Identification of efficacious RSV therapeutics has been hindered by the lack of a robust and appropriate primary assay for high-throughput screening (HTS). Here we report an HTS cascade that identified inhibitors of RSV replication using a robust RSV replicon luminescence-reporter assay for the primary campaign. The performance of the assay was consistent and reliable at scale, with Z′ of 0.55 ± 0.08 across 150 assay plates and signal-to-background ratios >40. The HTS assay was used to screen the AstraZeneca compound library of 1 million compounds at a single concentration of 10 µM. Hits specifically targeting the RSV replicon were determined using a series of hit generation assays. Compounds nonspecifically causing cell toxicity were removed, and hits were confirmed in live viral inhibition assays exhibiting greater physiological relevance than the primary assay. In summary, we developed a robust screening cascade that identified hit molecules that specifically targeted RSV replication.
•We established a robust RSV replicon assay for HTS.•This assay enabled large-scale screening for RSV replication inhibitors with low biocontainment requirement.•The RSV replicon assay was validated ...by multiple RSV replication inhibitors.•We further developed a multiplex assay that delivers both RSV replicon and cytotoxicity readouts.
Respiratory syncytial virus (RSV) drug discovery has been hindered by the lack of good chemistry starting points and would benefit from robust and convenient assays for high-throughput screening (HTS). In this paper, we present the development and optimization of a 384-well RSV replicon assay that enabled HTS for RSV replication inhibitors with a low bio-containment requirement. The established replicon assay was successfully implemented for high-throughput screening. A validation screen was performed which demonstrated high assay performance and reproducibility. Assay quality was further confirmed via demonstration of appropriate pharmacology for different classes of RSV replication tool inhibitors. RSV replicon and cytotoxicity assays were further developed into a multiplexed format that measured both inhibition of viral replication and cytotoxicity from the same well. This provided a time and cost efficient approach to support lead optimization. In summary, we have developed a robust RSV replicon assay to help expedite the discovery of novel RSV therapeutics.
Respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory tract infections in young children and other high-risk populations. RSV nucleoprotein (N) is essential for virus ...assembly and replication as part of the viral ribonucleoprotein (RNP) complex. RSV604 was a putative N inhibitor in phase 2 clinical trials whose molecular mechanism of action (MoA) was not well understood. This study investigated the cell line-dependent potency of RSV604 and demonstrated its direct binding to the N protein in vitro, providing the first evidence of direct target engagement for this class of inhibitors reported to date. The affinity of RSV604 N binding was not affected by RSV604 resistance mutations in the N protein. RSV604 engaged in two different MoAs in HeLa cells, inhibiting both RSV RNA synthesis and the infectivity of released virus. The lack of inhibition of viral RNA synthesis in some cell lines explained the cell-type-dependent potency of the inhibitor. RSV604 did not inhibit viral RNA synthesis in the RSV subgenomic replicon cells or in the cell-free RNP assay, suggesting that it might act prior to viral replication complex formation. RSV604 did not alter N protein localization in the infected cells. Taken together, these results provide new insights leading to an understanding of the MoAs of RSV604 and other similar N inhibitors.
Here we describe the rep gene, isolated from an environmental DNA library, which when transformed into Streptomyces species resulted in increased production of secondary metabolites and accelerated ...sporulation. We show that Streptomyces lividans strains bearing rep are particularly useful as expression hosts for heterologous antibiotic production.
•Three novel RSV inhibitor series were identified by RSV replicon HTS.•Each series involved a distinct mode of action and resistance.•Provided tool compounds to advance RSV antiviral discovery and ...viral replication research.
Respiratory Syncytial Virus (RSV) is a major cause of lower respiratory tract infections with no effective treatment available. Finding novel inhibitors of RSV is an important first step towards developing an efficacious RSV therapy. Here we report the characterization of three novel classes of RSV replication inhibitors identified through a high throughput RSV replicon screen of ∼1million compounds in the AstraZeneca compound collection. These inhibitors, cpd 1, 2, and 3, specifically targeted RSV and were not active against other viruses tested. Resistance selection in RSV A2 with cpd 1 identified escape viruses with mutations mapped to the RSV L protein, an RNA-dependent RNA polymerase (Y1631C and I1413T). Recombinant RSV containing the L Y1631C substitution conferred resistance towards cpd 1, suggesting that the RSV polymerase is the target of this inhibitor. Interestingly, cpd 3, a nucleoside analog, induced a single resistant mutation in the P protein (D231V), indicating a novel mode of action not previously reported. cpd 2 affected host cell cycle and no frequent mutation was isolated following resistance selection, suggesting its possible involvement of a host-targeted mechanism. Taken together, we have identified three novel RSV inhibitors with different modes of action, providing new chemistry starting points for the discovery and development of future RSV therapeutic treatment.
The enormous diversity of uncultured microorganisms in soil and other environments provides a potentially rich source of novel natural products, which is critically important for drug discovery ...efforts. Our investigators reported previously on the creation and screening of an Escherichia coli library containing soil DNA cloned and expressed in a bacterial artificial chromosome (BAC) vector. In that initial study, our group identified novel enzyme activities and a family of antibacterial small molecules encoded by soil DNA cloned and expressed in E. coli. To continue our pilot study of the utility and feasibility of this approach to natural product drug discovery, we have expanded our technology to include Streptomyces lividans and Pseudomonas putida as additional hosts with different expression capabilities, and herein we describe the tools we developed for transferring environmental libraries into all three expression hosts and screening for novel activities. These tools include derivatives of S. lividans that contain complete and unmarked deletions of the act and red endogenous pigment gene clusters, a derivative of P. putida that can accept environmental DNA vectors and integrate the heterologous DNA into the chromosome, and new BAC shuttle vectors for transferring large fragments of environmental DNA from E. coli to both S. lividans and P. putida by high-throughput conjugation. Finally, we used these tools to confirm that the three hosts have different expression capabilities for some known gene clusters. PUBLICATION ABSTRACT
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