Redirecting raltitrexed from cancer cell thymidylate synthase to Mycobacterium tuberculosis phosphopantetheinyl transferase.

There is a compelling need to find drugs active against Mycobacterium tuberculosis (Mtb). 4'-Phosphopantetheinyl transferase (PptT) is an essential enzyme in Mtb that has attracted interest as a potential drug target. We optimized a PptT assay, used it to screen 422,740 compounds, and identified raltitrexed, an antineoplastic antimetabolite, as the most potent PptT inhibitor yet reported. While trying unsuccessfully to improve raltitrexed's ability to kill Mtb and remove its ability to kill human cells, we learned three lessons that may help others developing antibiotics. First, binding of raltitrexed substantially changed the configuration of the PptT active site, complicating molecular modeling of analogs based on the unliganded crystal structure or the structure of cocrystals with inhibitors of another class. Second, minor changes in the raltitrexed molecule changed its target in Mtb from PptT to dihydrofolate reductase (DHFR). Third, the structure-activity relationship for over 800 raltitrexed analogs only became interpretable when we quantified and characterized the compounds' intrabacterial accumulation and transformation.

Identification of epidermal growth factor receptor-tyrosine kinase inhibitor targeting the VP1 pocket of human rhinovirus.

Screening a library of 1,200 preselected kinase inhibitors for anti-human rhinovirus 2 (HRV-2) activity in HeLa cells identified a class of epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKI) as effective virus blockers. These were based on the 4-anilinoquinazoline-7-oxypiperidine scaffold, with the most potent representative AZ5385 inhibiting the virus with EC50 of 0.35 µM. Several structurally related analogs confirmed activity in the low µM range, while interestingly, other TKIs targeting EGFR lacked anti-HRV-2 activity. To further probe this lack of association between antiviral activity and EGFR inhibition, we stained infected cells with antibodies specific for activated EGFR (Y1068) and did not observe a dependency on EGFR-TK activity. Instead, consecutive passages of HRV-2 in HeLa cells in the presence of a compound and subsequent nucleotide sequence analysis of resistant viral variants identified the S181T and T210A alterations in the major capsid VP1 protein, with both residues located in the vicinity of a known hydrophobic pocket on the viral capsid. Further characterization of the antiviral effects of AZ5385 showed a modest virus-inactivating (virucidal) activity, while anti-HRV-2 activity was still evident when the inhibitor was added as late as 10 h post infection. The RNA copy/infectivity ratio of HRV-2 propagated in AZ5385 presence was substantially higher than that of control HRV indicating that the compound preferentially targeted HRV progeny virions during their maturation in infected cells. Besides HRV, the compound showed anti-respiratory syncytial virus activity, which warrants its further studies as a candidate compound against viral respiratory infections.

Generating Selective Leads for Mer Kinase Inhibitors-Example of a Comprehensive Lead-Generation Strategy.

Mer is a member of the TAM (Tyro3, Axl, Mer) kinase family that has been associated with cancer progression, metastasis, and drug resistance. Their essential function in immune homeostasis has prompted an interest in their role as modulators of antitumor immune response in the tumor microenvironment. Here we illustrate the outcomes of an extensive lead-generation campaign for identification of Mer inhibitors, focusing on the results from concurrent, orthogonal high-throughput screening approaches. Data mining, HT (high-throughput), and DECL (DNA-encoded chemical library) screens offered means to evaluate large numbers of compounds. We discuss campaign strategy and screening outcomes, and exemplify series resulting from prioritization of hits that were identified. Concurrent execution of HT and DECL screening successfully yielded a large number of potent, selective, and novel starting points, covering a range of selectivity profiles across the TAM family members and modes of kinase binding, and offered excellent start points for lead development.

A High-Throughput Cellular Screening Assay for Small-Molecule Inhibitors and Activators of Cytoplasmic Dynein-1-Based Cargo Transport.

Cytoplasmic dynein-1 (hereafter dynein) is a six-subunit motor complex that transports a variety of cellular components and pathogens along microtubules. Dynein's cellular functions are only partially understood, and potent and specific small-molecule inhibitors and activators of this motor would be valuable for addressing this issue. It has also been hypothesized that an inhibitor of dynein-based transport could be used in antiviral or antimitotic therapy, whereas an activator could alleviate age-related neurodegenerative diseases by enhancing microtubule-based transport in axons. Here, we present the first high-throughput screening (HTS) assay capable of identifying both activators and inhibitors of dynein-based transport. This project is also the first collaborative screening report from the Medical Research Council and AstraZeneca agreement to form the UK Centre for Lead Discovery. A cellular imaging assay was used, involving chemically controlled recruitment of activated dynein complexes to peroxisomes. Such a system has the potential to identify molecules that affect multiple aspects of dynein biology in vivo. Following optimization of key parameters, the assay was developed in a 384-well format with semiautomated liquid handling and image acquisition. Testing of more than 500,000 compounds identified both inhibitors and activators of dynein-based transport in multiple chemical series. Additional analysis indicated that many of the identified compounds do not affect the integrity of the microtubule cytoskeleton and are therefore candidates to directly target the transport machinery.

A High-Throughput Screening Triage Workflow to Authenticate a Novel Series of PFKFB3 Inhibitors.

A high-throughput screen (HTS) of human 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) resulted in several series of compounds with the potential for further optimization. Informatics was used to identify active chemotypes with lead-like profiles and remove compounds that commonly occurred as actives in other HTS screens. The activities were confirmed with IC50 measurements from two orthogonal assay technologies, and further analysis of the Hill slopes and comparison of the ratio of IC50 values at 10 times the enzyme concentration were used to identify artifact compounds. Several series of compounds were rejected as they had both high slopes and poor ratios. A small number of compounds representing the different leading series were assessed using isothermal titration calorimetry, and the X-ray crystal structure of the complex with PFKFB3 was solved. The orthogonal assay technology and isothermal calorimetry were demonstrated to be unreliable in identifying false-positive compounds in this case. Presented here is the discovery of the dihydropyrrolopyrimidinone series of compounds as active and novel inhibitors of PFKFB3, shown by X-ray crystallography to bind to the adenosine triphosphate site. The crystal structures of this series also reveal it is possible to flip the binding mode of the compounds, and the alternative orientation can be driven by a sigma-hole interaction between an aromatic chlorine atom and a backbone carbonyl oxygen. These novel inhibitors will enable studies to explore the role of PFKFB3 in driving the glycolytic phenotype of tumors.

Identification and Characterization of Dual Inhibitors of the USP25/28 Deubiquitinating Enzyme Subfamily.

The ubiquitin proteasome system is widely postulated to be a new and important field of drug discovery for the future, with the ubiquitin specific proteases (USPs) representing one of the more attractive target classes within the area. Many USPs have been linked to critical axes for therapeutic intervention, and the finding that USP28 is required for c-Myc stability suggests that USP28 inhibition may represent a novel approach to targeting this so far undruggable oncogene. Here, we describe the discovery of the first reported inhibitors of USP28, which we demonstrate are able to bind to and inhibit USP28, and while displaying a dual activity against the closest homologue USP25, these inhibitors show a high degree of selectivity over other deubiquitinases (DUBs). The utility of these compounds as valuable probes to investigate and further explore cellular DUB biology is highlighted by the demonstration of target engagement against both USP25 and USP28 in cells. Furthermore, we demonstrate that these inhibitors are able to elicit modulation of both the total levels and the half-life of the c-Myc oncoprotein in cells and also induce apoptosis and loss of cell viability in a range of cancer cell lines. We however observed a narrow therapeutic index compared to a panel of tissue-matched normal cell lines. Thus, it is hoped that these probes and data presented herein will further advance our understanding of the biology and tractability of DUBs as potential future therapeutic targets.

Discovery of Imidazo[1,2-a]pyridine Ethers and Squaramides as Selective and Potent Inhibitors of Mycobacterial Adenosine Triphosphate (ATP) Synthesis.

The approval of bedaquiline to treat tuberculosis has validated adenosine triphosphate (ATP) synthase as an attractive target to kill Mycobacterium tuberculosis (Mtb). Herein, we report the discovery of two diverse lead series imidazo[1,2-a]pyridine ethers (IPE) and squaramides (SQA) as inhibitors of mycobacterial ATP synthesis. Through medicinal chemistry exploration, we established a robust structure-activity relationship of these two scaffolds, resulting in nanomolar potencies in an ATP synthesis inhibition assay. A biochemical deconvolution cascade suggested cytochrome c oxidase as the potential target of IPE class of molecules, whereas characterization of spontaneous resistant mutants of SQAs unambiguously identified ATP synthase as its molecular target. Absence of cross resistance against bedaquiline resistant mutants suggested a different binding site for SQAs on ATP synthase. Furthermore, SQAs were found to be noncytotoxic and demonstrated efficacy in a mouse model of tuberculosis infection.

Discovery of a novel allosteric inhibitor-binding site in ERK5: comparison with the canonical kinase hinge ATP-binding site.

MAP kinases act as an integration point for multiple biochemical signals and are involved in a wide variety of cellular processes such as proliferation, differentiation, regulation of transcription and development. As a member of the MAP kinase family, ERK5 (MAPK7) is involved in the downstream signalling pathways of various cell-surface receptors, including receptor tyrosine kinases and G protein-coupled receptors. In the current study, five structures of the ERK5 kinase domain co-crystallized with ERK5 inhibitors are reported. Interestingly, three of the compounds bind at a novel allosteric binding site in ERK5, while the other two bind at the typical ATP-binding site. Binding of inhibitors at the allosteric site is accompanied by displacement of the P-loop into the ATP-binding site and is shown to be ATP-competitive in an enzymatic assay of ERK5 kinase activity. Kinase selectivity data show that the most potent allosteric inhibitor exhibits superior kinase selectivity compared with the two inhibitors that bind at the canonical ATP-binding site. An analysis of these structures and comparison with both a previously published ERK5-inhibitor complex structure (PDB entry 4b99) and the structures of three other kinases (CDK2, ITK and MEK) in complex with allosteric inhibitors are presented.

A novel high-throughput cell-based assay aimed at identifying inhibitors of DNA metabolism in bacteria.

Bacterial biosensor strains can be useful tools for the discovery and characterization of antibacterial compounds. A plasmid-based reporter vector containing a transcriptional fusion between the recA promoter and green fluorescence protein gene was introduced into an Escherichia coli ΔtolC strain to create a biosensor strain that selectively senses inhibitors of DNA metabolism via the SOS response. The strain was used to develop a high-throughput assay to identify new inhibitors of DNA metabolism. Screening of the AstraZeneca compound library with this strain identified known inhibitors of DNA metabolism, as well as novel chemotypes. The cellular target of one novel series was elucidated as DNA gyrase through genetic characterization of laboratory-generated resistant mutants followed by 50% inhibitory concentration measurements in a DNA gyrase activity assay. These studies validated the use of this antibiotic biosensor strain to identify novel selective inhibitors of DNA metabolism by high-throughput screening.

Potent, selective small molecule inhibitors of type III phosphatidylinositol-4-kinase α- but not ß-inhibit the phosphatidylinositol signaling cascade and cancer cell proliferation.

Two series of inhibitors of type III phosphatidylinositol-4-kinase were identified by high throughput screening and optimised to derive probe compounds that independently and selectively inhibit the α- and the ß-isoforms with no significant activity towards related kinases in the pathway. In a cellular environment, inhibition of the α- but not the ß-subtype led to a reduction in phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate concentration, causing inhibition of inositol-1-phosphate formation and inhibition of proliferation in a panel of cancer cell lines.