Design, Synthesis, and Biological Evaluation of Quinazolin-4-one-Based Hydroxamic Acids as Dual PI3K/HDAC Inhibitors.

A series of quinazolin-4-one based hydroxamic acids was rationally designed and synthesized as novel dual PI3K/HDAC inhibitors by incorporating an HDAC pharmacophore into a PI3K inhibitor (Idelalisib) via an optimized linker. Several of these dual inhibitors were highly potent (IC50 < 10 nM) and selective against PI3Kγ, δ and HDAC6 enzymes and exhibited good antiproliferative activity against multiple cancer cell lines. The lead compound 48c, induced necrosis in several mutant and FLT3-resistant AML cell lines and primary blasts from AML patients, while showing no cytotoxicity against normal PBMCs, NIH3T3, and HEK293 cells. Target engagement of PI3Kδ and HDAC6 by 48c was demonstrated in MV411 cells using the cellular thermal shift assay (CETSA). Compound 48c showed good pharmacokinetics properties in mice via intraperitoneal (ip) administration and provides a means to examine the biological effects of inhibiting these two important enzymes with a single molecule, either in vitro or in vivo.

Computer-Aided Discovery and Characterization of Novel Ebola Virus Inhibitors.

The Ebola virus (EBOV) causes severe human infection that lacks effective treatment. A recent screen identified a series of compounds that block EBOV-like particle entry into human cells. Using data from this screen, quantitative structure-activity relationship models were built and employed for virtual screening of a ∼17 million compound library. Experimental testing of 102 hits yielded 14 compounds with IC50 values under 10 µM, including several sub-micromolar inhibitors, and more than 10-fold selectivity against host cytotoxicity. These confirmed hits include FDA-approved drugs and clinical candidates with non-antiviral indications, as well as compounds with novel scaffolds and no previously known bioactivity. Five selected hits inhibited BSL-4 live-EBOV infection in a dose-dependent manner, including vindesine (0.34 µM). Additional studies of these novel anti-EBOV compounds revealed their mechanisms of action, including the inhibition of NPC1 protein, cathepsin B/L, and lysosomal function. Compounds identified in this study are among the most potent and well-characterized anti-EBOV inhibitors reported to date.

Novel lead structures with both Plasmodium falciparum gametocytocidal and asexual blood stage activity identified from high throughput compound screening.

BACKGROUND: Blocking malaria transmission is an important step in eradicating malaria. In the field, transmission requires the production of sexual stage Plasmodium parasites, called gametocytes, which are not effectively killed by the commonly used anti-malarials allowing individuals to remain infectious after clearance of asexual parasites. METHODS: To identify new gametocytocidal compounds, a library of 45,056 compounds with diverse structures was screened using a high throughput gametocyte viability assay. The characteristics of active hits were further evaluated against asexual stage parasites in a growth inhibition assay. Their cytotoxicity were tested against mammalian cells in a cytotoxicity assay. The chemical scaffold similarity of active hits were studied using scaffold cluster analysis. RESULTS: A set of 23 compounds were identified and further confirmed for their activity against gametocytes. All the 23 confirmed compounds possess dual-activities against both gametocytes responsible for human to mosquito transmission and asexual parasites that cause the clinical symptoms. Three of these compounds were fourfold more active against gametocytes than asexual parasites. Further cheminformatic analysis revealed three sets of novel scaffolds, including highly selective 4-1H-pyrazol-5-yl piperidine analogs. CONCLUSIONS: This study revealed important new structural scaffolds that can be used as starting points for dual activity anti-malarial drug development.

Rapid countermeasure discovery against Francisella tularensis based on a metabolic network reconstruction.

In the future, we may be faced with the need to provide treatment for an emergent biological threat against which existing vaccines and drugs have limited efficacy or availability. To prepare for this eventuality, our objective was to use a metabolic network-based approach to rapidly identify potential drug targets and prospectively screen and validate novel small-molecule antimicrobials. Our target organism was the fully virulent Francisella tularensis subspecies tularensis Schu S4 strain, a highly infectious intracellular pathogen that is the causative agent of tularemia and is classified as a category A biological agent by the Centers for Disease Control and Prevention. We proceeded with a staggered computational and experimental workflow that used a strain-specific metabolic network model, homology modeling and X-ray crystallography of protein targets, and ligand- and structure-based drug design. Selected compounds were subsequently filtered based on physiological-based pharmacokinetic modeling, and we selected a final set of 40 compounds for experimental validation of antimicrobial activity. We began screening these compounds in whole bacterial cell-based assays in biosafety level 3 facilities in the 20th week of the study and completed the screens within 12 weeks. Six compounds showed significant growth inhibition of F. tularensis, and we determined their respective minimum inhibitory concentrations and mammalian cell cytotoxicities. The most promising compound had a low molecular weight, was non-toxic, and abolished bacterial growth at 13 µM, with putative activity against pantetheine-phosphate adenylyltransferase, an enzyme involved in the biosynthesis of coenzyme A, encoded by gene coaD. The novel antimicrobial compounds identified in this study serve as starting points for lead optimization, animal testing, and drug development against tularemia. Our integrated in silico/in vitro approach had an overall 15% success rate in terms of active versus tested compounds over an elapsed time period of 32 weeks, from pathogen strain identification to selection and validation of novel antimicrobial compounds.

Exploring polypharmacology using a ROCS-based target fishing approach.

Polypharmacology has emerged as a new theme in drug discovery. In this paper, we studied polypharmacology using a ligand-based target fishing (LBTF) protocol. To implement the protocol, we first generated a chemogenomic database that links individual protein targets with a specified set of drugs or target representatives. Target profiles were then generated for a given query molecule by computing maximal shape/chemistry overlap between the query molecule and the drug sets assigned to each protein target. The overlap was computed using the program ROCS (Rapid Overlay of Chemical Structures). We validated this approach using the Directory of Useful Decoys (DUD). DUD contains 2950 active compounds, each with 36 property-matched decoys, against 40 protein targets. We chose a set of known drugs to represent each DUD target, and we carried out ligand-based virtual screens using data sets of DUD actives seeded into DUD decoys for each target. We computed Receiver Operator Characteristic (ROC) curves and associated area under the curve (AUC) values. For the majority of targets studied, the AUC values were significantly better than for the case of a random selection of compounds. In a second test, the method successfully identified off-targets for drugs such as rimantadine, propranolol, and domperidone that were consistent with those identified by recent experiments. The results from our ROCS-based target fishing approach are promising and have potential application in drug repurposing for single and multiple targets, identifying targets for orphan compounds, and adverse effect prediction.

Ion channel screening plates: design, construction, and maintenance.

Ion channels have provided a diverse set of therapeutic targets across all areas of the pharmaceutical industry. Many companies are pursuing this unique class of targets for areas of unmet medical need such as neuropathic and inflammatory pains. In the past, focused library screening sets had been designed for CNS and kinase targets. Our investigations were aimed at creating a similar dynamic screening set enriched for compounds targeting ion channels to aid screening efforts of this important class of targets. The key advantages of this approach for ion channel targets would be: (1) to identify tool compounds for novel targets and assist in assay validation, (2) to serve as a focused screen for non-384-well adaptable targets, and (3) to jump start a particular program, that is, catch-up to competition for validated, well-known targets.

Ion channel screening.

Ion channels are attractive targets for drug discovery with recent estimates indicating that voltage and ligand-gated channels account for the third and fourth largest gene families represented in company portfolios after the G protein coupled and nuclear hormone receptor families. A historical limitation on ion channel targeted drug discovery in the form of the extremely low throughput nature of the gold standard assay for assessing functional activity, patch clamp electrophysiology in mammalian cells, has been overcome by the implementation of multi-well plate format cell-based screening strategies for ion channels. These have taken advantage of various approaches to monitor ion flux or membrane potential using radioactive, non-radioactive, spectroscopic and fluorescence measurements and have significantly impacted both high-throughput screening and lead optimization efforts. In addition, major advances have been made in the development of automated electrophysiological platforms to increase capacity for cell-based screening using formats aimed at recapitulating the gold standard assay. This review addresses the options available for cell-based screening of ion channels with examples of their utility and presents case studies on the successful implementation of high-throughput screening campaigns for a ligand-gated ion channel using a fluorescent calcium indicator, and a voltage-gated ion channel using a fluorescent membrane potential sensitive dye.