High cancer drug development attrition rates have provoked considerable debate about whether the two-dimensional tumor growth inhibition high-throughput screening assays used in pre-clinical lead discovery adequately reflect solid tumor complexity. We used automated high-content screening image acquisition and analysis methods to compare fluorescent drug uptake, accumulation, and distribution in Cal33 and FaDu head and neck cancer (HNC) monolayer and multicellular tumor spheroid (MCTS) models. Ellipticine, idarubicin, daunorubicin, and doxorubicin were studied because of their fluorescent properties and broad anti-tumor activities. HNC MCTSs were generated in 384-well ultra-low attachment plates where compound exposure, image acquisition, and analysis could be performed in situ. Fluorescent drug accumulation in Cal33 monolayer and MCTS cultures was linear with respect to concentration, and appeared to achieve steady-state levels within 10-15 min of drug exposure, which were maintained through 30-45 min. Drug accumulation in monolayers was independent of cell number and/or density, and every cell achieved uniform drug concentrations. In MCTSs, however, drug accumulation increased as the number of cells and sizes of the MCTSs became bigger. Drugs exhibited restricted penetration and distribution gradients, accumulating preferentially in cells in the outer layers of MCTSs relative to those in the inner cores. Cal33 monolayers were 6-, 20-, 10-, and 16-fold more sensitive than MCTSs to growth inhibition by ellipticine, idarubicin, daunorubicin, and doxorubicin, respectively. In Cal33 MCTSs exposed to ellipticine or doxorubicin for 24 h, MCTSs were smaller and although they still exhibited drug penetration and distribution gradients, the fluorescent intensity difference between outer and inner cells was reduced. After a 24 h exposure, both drugs had penetrated throughout FaDu MCTSs, consistent with drug-induced death of peripheral cell layers enhancing drug penetration. The increased resistance of MCTS cultures and their ability to recapitulate drug penetration and distribution gradients argues strongly for the deployment of these more physiological models in cancer lead discovery. MCTSs have the potential to enhance the correlation between in vitro potencies and in vivo efficacy, and ultimately may lead to improved cancer drug approval rates.

The dictyostatins are a promising class of potential anticancer drugs because they are powerful microtubule stabilizing agents, but the complexity of their chemical structures is a severe impediment to their further development. Based on both synthetic and medicinal chemistry analyses, 25,26-dihydro-16-desmethyldictyostatin and its C6 epimer were chosen as potentially potent yet accessible dictyostatin analogs, and three new syntheses were developed. A relatively classical synthesis involving vinyllithium addition and macrocyclization gave way to a newer and more practical approach based on esterification and ring-closing metathesis reaction. Finally, aspects of these two approaches were combined to provide a third new synthesis based on esterification and Nozaki-Hiyama-Kishi reaction. This was used to prepare the target dihydro analogs and the natural product. All of the syntheses are streamlined because of their high convergence. The work provided several new analogs of dictyostatin including a truncated macrolactone and a C10 E-alkene, which were 400-fold and 50-fold less active than (−)-dictyostatin. In contrast, the targeted 25,26-dihydro-16-desmethyldictyostatin analogs retained almost complete activity in preliminary biological assays.

SARS-CoV-2 (2019-nCoV) is the pathogenic coronavirus responsible for the global pandemic of COVID-19 disease. The Spike (S) protein of SARS-CoV-2 attaches to host lung epithelial cells through the cell surface receptor ACE2, a process dependent on host proteases including TMPRSS2. Here, we identify small molecules that reduce surface expression of TMPRSS2 using a library of 2,560 FDA-approved or current clinical trial compounds. We identify homoharringtonine and halofuginone as the most attractive agents, reducing endogenous TMPRSS2 expression at sub-micromolar concentrations. These effects appear to be mediated by a drug-induced alteration in TMPRSS2 protein stability. We further demonstrate that halofuginone modulates TMPRSS2 levels through proteasomal-mediated degradation that involves the E3 ubiquitin ligase component DDB1- and CUL4-associated factor 1 (DCAF1). Finally, cells exposed to homoharringtonine and halofuginone, at concentrations of drug known to be achievable in human plasma, demonstrate marked resistance to SARS-CoV-2 infection in both live and pseudoviral in vitro models. Given the safety and pharmacokinetic data already available for the compounds identified in our screen, these results should help expedite the rational design of human clinical trials designed to combat active COVID-19 infection.

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Signal transducer and activator of transcription factor 3 (STAT3) is hyperactivated in head and neck squamous cell carcinomas (HNSCC). Cumulative evidence indicates that IL-6 production by HNSCC cells and/or stromal cells in the tumor microenvironment activates STAT3 and contributes to tumor progression and drug resistance. A library of 94,491 compounds from the Molecular Library Screening Center Network (MLSCN) was screened for the ability to inhibit interleukin-6 (IL-6)-induced pSTAT3 activation. For contractual reasons, the primary high-content screening (HCS) campaign was conducted over several months in 3 distinct phases; 1,068 (1.1%) primary HCS actives remained after cytotoxic or fluorescent outliers were eliminated. One thousand one hundred eighty-seven compounds were cherry-picked for confirmation; actives identified in the primary HCS and compounds selected by a structural similarity search of the remaining MLSCN library using hits identified in phases I and II of the screen. Actives were confirmed in pSTAT3 IC 50 assays, and an IFNc-induced pSTAT1 activation assay was used to prioritize selective inhibitors of STAT3 activation that would not inhibit STAT1 tumor suppressor functions. Two hundred three concentration-dependent inhibitors of IL-6-induced pSTAT3 activation were identified and 89 of these also produced IC 50 s against IFN-c-induced pSTAT1 activation. Forty-nine compounds met our hit criteria: they reproducibly inhibited IL-6-induced pSTAT3 activation by ‡70% at 20 lM; their pSTAT3 activation IC 50 s were £25 lM; they were ‡2-fold selective for pSTAT3 inhibition over pSTAT1 inhibition; a cross target query of PubChem indicated that they were not biologically promiscuous; and they were ‡90% pure. Twenty-six chemically tractable hits that passed filters for nuisance compounds and had acceptable drug-like and ADME-Tox properties by computational evaluation were purchased for characterization. The hit structures were distributed among 5 clusters and 8 singletons. Twenty-four compounds inhibited IL-6-induced pSTAT3 activation with IC 50 s £20 lM and 13 were ‡3-fold selective versus inhibition of pSTAT1 activation. Eighteen hits inhibited the growth of HNSCC cell lines with average IC 50 s £ 20 lM. Four chemical series were progressed into lead optimization: the guanidinoquinazolines, the triazolothiadiazines, the amino alcohols, and an oxazole-piperazine singleton.