High-Throughput Screening and Triage Assays Identify Small Molecules Targeting c-MYC in Cancer Cells.

While c-MYC is well established as a proto-oncogene, its structure and function as a transcription factor have made c-MYC a difficult therapeutic target. To identify small-molecule inhibitors targeting c-MYC for anticancer therapy, we designed a high-throughput screening (HTS) strategy utilizing cellular assays. The novel approach for the HTS was based on the detection of cellular c-MYC protein, with active molecules defined as those that specifically decreased c-MYC protein levels in cancer cells. The assay was based on a dual antibody detection system using Förster/fluorescence resonance energy transfer (FRET) and was utilized to detect endogenous c-MYC protein in the MYC amplified cancer cell lines DMS273 and Colo320 HSR. The assays were miniaturized to 1536-well plate format and utilized to screen the GlaxoSmithKline small-molecule collection of approximately 2 million compounds. In addition to the HTS assay, follow-up assays were developed and used to triage and qualify compounds. Two cellular assays used to eliminate false-positive compounds from the initially selected HTS hits were (1) a cellular toxicity assay and (2) an unstable protein reporter assay. Three positive selection assays were subsequently used to qualify compounds: (1) 384-well cell cycle flow cytometry, (2) 384-well cell growth, and (3) c-MYC gene signature reverse transcription quantitative PCR (RT-qPCR). The HTS and follow-up assays successfully identified three compounds that specifically decreased c-MYC protein levels in cancer cells and phenocopied c-MYC siRNA in terms of cell growth inhibition and gene signatures. The HTS, triage, and three compounds identified are described.

Scaffold-Based Analytics: Enabling Hit-to-Lead Decisions by Visualizing Chemical Series Linked across Large Datasets.

We present a method for visualizing and navigating large screening datasets while also taking into account their activities and properties. Our approach is to annotate the data with all possible scaffolds contained within each molecule. We have developed a Spotfire visualization, coupled to a fuzzy clustering approach based on the scaffold decomposition of the screening deck, used to drive the hit triage process. Progression decisions can be made using aggregate scaffold parameters and data from multiple datasets merged at the scaffold level. This visualization reveals overlaps that help prioritize hits, highlight tractable series, and posit ways to combine aspects of multiple hits. The structure-activity relationship of a large and complex hit is automatically mapped onto all constituent scaffolds making it possible to navigate, via any shared scaffold, to all related hits. This scaffold "walking" helps address bias toward a handful of potent and ligand-efficient molecules at the expense of coverage of chemical space. We consider two scaffold generation methods and explored their similarities and differences both qualitatively and quantitatively. The workflow of a Spotfire visualization used in combination with fuzzy clustering and structure annotation provides an intuitive view of large and diverse screening datasets. This allows teams to effortlessly navigate between structurally related molecules and enriches the population of leads considered and progressed in a manner complementary to established approaches.

Novel KV7 ion channel openers for the treatment of epilepsy and implications for detrusor tissue contraction.

Neuronal voltage-gated potassium channels, KV7s, are the molecular mediators of the M current and regulate membrane excitability in the central and peripheral neuronal systems. Herein, we report novel small molecule KV7 openers that demonstrate anti-seizure activities in electroshock and pentylenetetrazol-induced seizure models without influencing Rotarod readouts in mice. The anti-seizure activity was determined to be proportional to the unbound concentration in the brain. KV7 channels are also expressed in the bladder smooth muscle (detrusor) and activation of these channels may cause localized undesired effects. Therefore, the impact of individual KV7 isoforms was investigated in human detrusor tissue using a panel of KV7 openers with distinct activity profiles among KV7 isoforms. KCNQ4 and KCNQ5 mRNA were highly expressed in detrusor tissue, yet a compound that has significantly reduced activity on homomeric KV7.4 did not reduce detrusor contraction. This may suggest that the homomeric KV7.4 channel plays a less significant role in bladder contraction and further investigation is needed.

Coal mine dust lung disease in the modern era.

Coal workers' pneumoconiosis (CWP), as part of the spectrum of coal mine dust lung disease (CMDLD), is a preventable but incurable lung disease that can be complicated by respiratory failure and death. Recent increases in coal production from the financial incentive of economic growth lead to higher respirable coal and quartz dust levels, often associated with mechanization of longwall coal mining. In Australia, the observed increase in the number of new CWP diagnoses since the year 2000 has necessitated a review of recommended respirable dust exposure limits, where exposure limits and monitoring protocols should ideally be standardized. Evidence that considers the regulation of engineering dust controls in the mines is lacking even in high-income countries, despite this being the primary preventative measure. Also, it is a global public health priority for at-risk miners to be systemically screened to detect early changes of CWP and to include confirmed patients within a central registry; a task limited by financial constraints in less developed countries. Characteristic X-ray changes are usually categorized using the International Labour Office classification, although future evaluation by low-dose HRCT) chest scanning may allow for CWP detection and thus avoidance of further exposure, at an earlier stage. Preclinical animal and human organoid-based models are required to explore potential re-purposing of anti-fibrotic and related agents with potential efficacy. Epidemiological patterns and the assessment of molecular and genetic biomarkers may further enhance our capacity to identify susceptible individuals to the inhalation of coal dust in the modern era.

Discovery and Characterization of a Class of Pyrazole Inhibitors of Bacterial Undecaprenyl Pyrophosphate Synthase.

Undecaprenyl pyrophosphate synthase (UppS) is an essential enzyme in bacterial cell wall synthesis. Here we report the discovery of Staphylococcus aureus UppS inhibitors from an Encoded Library Technology screen and demonstrate binding to the hydrophobic substrate site through cocrystallography studies. The use of bacterial strains with regulated uppS expression and inhibitor resistant mutant studies confirmed that the whole cell activity was the result of UppS inhibition, validating UppS as a druggable antibacterial target.

New IDH1 mutant inhibitors for treatment of acute myeloid leukemia.

Neomorphic mutations in isocitrate dehydrogenase 1 (IDH1) are driver mutations in acute myeloid leukemia (AML) and other cancers. We report the development of new allosteric inhibitors of mutant IDH1. Crystallographic and biochemical results demonstrated that compounds of this chemical series bind to an allosteric site and lock the enzyme in a catalytically inactive conformation, thereby enabling inhibition of different clinically relevant IDH1 mutants. Treatment of IDH1 mutant primary AML cells uniformly led to a decrease in intracellular 2-HG, abrogation of the myeloid differentiation block and induction of granulocytic differentiation at the level of leukemic blasts and more immature stem-like cells, in vitro and in vivo. Molecularly, treatment with the inhibitors led to a reversal of the DNA cytosine hypermethylation patterns caused by mutant IDH1 in the cells of individuals with AML. Our study provides proof of concept for the molecular and biological activity of novel allosteric inhibitors for targeting different mutant forms of IDH1 in leukemia.

Structure-based design of potent and selective 3-phosphoinositide-dependent kinase-1 (PDK1) inhibitors.

Phosphoinositide-dependent protein kinase-1(PDK1) is a master regulator of the AGC family of kinases and an integral component of the PI3K/AKT/mTOR pathway. As this pathway is among the most commonly deregulated across all cancers, a selective inhibitor of PDK1 might have utility as an anticancer agent. Herein we describe our lead optimization of compound 1 toward highly potent and selective PDK1 inhibitors via a structure-based design strategy. The most potent and selective inhibitors demonstrated submicromolar activity as measured by inhibition of phosphorylation of PDK1 substrates as well as antiproliferative activity against a subset of AML cell lines. In addition, reduction of phosphorylation of PDK1 substrates was demonstrated in vivo in mice bearing OCl-AML2 xenografts. These observations demonstrate the utility of these molecules as tools to further delineate the biology of PDK1 and the potential pharmacological uses of a PDK1 inhibitor.